Methods and compositions for treating conditions

ABSTRACT

The invention relates to compositions comprising of SEQ NO: 1-244, 248-249, and any homologs, analogs, and fragments thereof. Such compositions can be used to treat, prevent, and modulate pain, inflammation, and metabolic processes in various organisms including plants and animals. Such compositions can be formulated with an acceptable pharmaceutical excipient for administration to a human or a plant. The compositions can be administered topically or for systemic use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and is a Continuation Applicationof U.S. Continuation application Ser. No. 12/582,008, filed Oct. 20,2009 which claims the benefit of U.S. Utility application Ser. No.11/287,157, filed Nov. 25, 2005 which claims the benefit of ProvisionalPatent Application Nos. 60/630,880, filed Nov. 24, 2004, 60/652,287,filed Feb. 10, 2005, and 60/658,859 filed Mar. 4, 2005, which are herebyincorporated by reference for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 17, 2011, isnamed 32051-701.302Seqlist.txt and is 100 Kilobytes in size.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 17, 2011, isnamed 32051-701.302Seqlist.txt and is 100 Kilobytes in size.

BACKGROUND

Pain is a condition that affects everyone at some point in his lifetime.The pain signaling pathway can be modulated by endogenous and syntheticopioid peptides and by small molecules. Pain is induced by multipletypes of stimuli such as temperature and tissue damage suggesting thatthese pathways have at least one common component. Currently, thetherapeutic choice in the management of severe acute and chronic pain isat the plasma membrane site, where the opioid peptides act as agonistsfor different opioid receptors.

As the world is experiencing an increase in life expectancy andpopulation size, there is a significant need to identify newcompositions and methods to treat and/or prevent different physiologicalconditions associated with pain, inflammation, thermoregulation, andother mitochondria-associated condition in both animals and plants.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

SUMMARY OF THE INVENTION

The invention herein involves compositions comprising, consistingessentially of, or consisting of a polypeptide of the invention or ahomolog, analog, mimetic, salt, prodrug, metabolite, or fragmentthereof. In some embodiments, a polypeptide comprises, consistsessentially of, or consists of one or more amino acid sequences of SEQID NOs: 1-244, 248-249 or a reverse sequence of SEQ ID NOs: 1-244,248-249. For example, in some embodiments, a composition comprises apolypeptide having amino acid sequence of SEQ ID NO: 1-14 or 50-244,248-249, or SEQ ID NO: 1 or 2, or SEQ ID NO: 1. In some embodiments, acomposition comprises a polypeptide having an amino acid sequence whichis the reverse of SEQ ID NO: 1. The invention herein also contemplateshomologs, analogs, mimetics, salts, prodrugs, metabolites, and fragmentsof the above polypeptides and compositions comprising the same.

The compositions herein can be used to modulate, prevent, or treat pain,inflammation, infections (e.g., bacterial fungi, viruses, etc.), andmetabolic processes or conditions in an organism (plant or animal).Examples of metabolic conditions include, but are not limited to, pain,wound healing, inflammation, heat production, fever, homeothermy,breakdown of triglycerides, glycolysis, Krebs cycle, fermentation,photosynthesis, metabolic rate, biotic and abiotic stress, secretions,oxidative stress, stress, neoplastic growth, skin condition,cardiovascular conditions, neurological and neurodegenerativeconditions, mental and behavioral disorders. Such processes orconditions can occur in a cell, group of cells, or an entire organism.

The compositions herein can be used for modulating, preventing, treatingcondition(s) in organisms. Such organisms can be animals and/or plants.

In some embodiments, the compositions herein (e.g., a compositioncomprising a polypeptide of SEQ ID NOs: 1-244, 248-249, or morepreferably SEQ ID NO: 1 are used to modulate or treat pain, such asnociceptive (non-chronic) pain, neuropathic (chronic) pain, idiopathicpain, headaches, low back pain, cancer pain, arthritis pain, sprains,bone fractures, pain resulting from burns, pain associated with bumps,pain associated with bruises, inflammatory pain (e.g., from an infectionor arthritic disorder), pain from obstructions, myofascial pain, painfrom nerve trauma (e.g., dystrophy/causalgia), phantom limb pain,entrapment neuropathy (e.g., carpal tunnel syndrome), and peripheralneuropathy.

Preferably, a composition comprising SEQ ID NO: 1 or a salt, metabolite,or prodrug thereof is administered to an animal to treat pain. Such paincan be non-chronic pain, neuropathic pain, or idiopathic pain. It isfurther contemplated a compositions comprising a polypeptide describedherein (e.g., SEQ ID NO: 1) is co-administered with one or more otherpain relief medications. For example, a polypeptide described herein,such as SEQ ID NO: 1 can be administered simultaneously with,co-formulated with, or administered in the same therapy as a painreliever selected from the group consisting of small molecules (e.g.,non-narcotic and narcotic analgesics) and peptide opioids.

In some embodiments, the compositions herein (e.g., a compositioncomprising a polypeptide comprising, consisting essentially, orconsisting of SEQ ID NOs: 1-244, 248-249, or more preferably SEQ IDNO: 1) are used to module or treat inflammatory conditions that may ormay not cause pain. Such conditions may show one or more of thefollowing symptoms: redness, heat, tenderness and swelling. Examples ofsuch conditions include, but are not limited to, chronic inflammatorydiseases, such as rheumatoid arthritis, inflammatory bowel disease,systemic lupus erythematosus, multiple sclerosis, and type I and IIdiabetes, asthma, and inflammatory diseases of the central nervoussystem such as multiple sclerosis, abscess, meningitis, encephalitis andvasculitis.

In some embodiments, the compositions herein (e.g., a compositioncomprising a polypeptide comprising, consisting essentially, orconsisting of SEQ ID NOs: 1-244, 248-249, or more preferably SEQ IDNO: 1) are used to modulate or treat cardiovascular conditions. Examplesof cardiovascular conditions associated with pain and/or inflammationinclude, but are not limited to, angina, arrhythmia, high bloodpressure, stroke, congestive heart failure, atherosclerosis, peripheralartery diseases, high cholesterol levels, and heart attacks.

In some embodiments, the compositions herein (e.g., a compositioncomprising a polypeptide comprising, consisting essentially, orconsisting of SEQ ID NOs: 1-244, 248-249, or more preferably SEQ IDNO: 1) are used to modulate or treat a neurological or neurodegenerativecondition or a mental or behavioral disorder. Examples of neurologicalconditions associated with pain and/or inflammation include, but are notlimited to, Alzheimer's disease, amnesia, Aicardi syndrome, amyotrophiclateral sclerosis (Lou Gehrig's disease), anencephaly, anxiety, aphasia,arachnoiditis, Arnold Chiari malformation, attention deficit syndrome,autism, Batten disease, Bell's Palsy, bipolar syndrome, brachial plexusinjury, brain injury, brain tumors, childhood depresses ion,Charcol-Marie tooth disease, depression, dystonia, dyslexia,encephalitis, epilepsy, essential tremor, Guillain-Barre syndrome,hydrocephalus, hyperhidrosis, Krabbes disease, learning disabilities,leukodystrophy, meningitis, Moebius syndrome, multiple sclerosis,muscular dystrophy, Parkinson's disease, peripheral neuropathy,obsessive compulsive disorder, postural orthostatic tachycardiasyndrome, progressive supranuclear palsy, prosopagnosia, schizophrenia,shingles, Shy-Drager syndrome, spasmodic torticollis, spina bifida,spinal muscular atrophy, stiff man syndrome, synesthesia, syringomyelia,thoracic outlet syndrome, tourette syndrome, toxoplasmosis, andtrigeminal neurolagia.

Examples of mental and behavioral disorders include, but are not limitedto, anxiety disorder, panic disorder, obsessive-compulsive disorder,post-traumatic stress disorder, social phobia (or social anxietydisorder), specific phobias, and generalized anxiety disorder. Any ofthe above conditions can also be accompanied by or manifested by otherconditions such as depression, drug abuse, or alcoholism.

In some embodiments, the compositions herein are used to treat feverthat occurs with many different conditions such as inflammation andinfectious diseases.

In some embodiments, the compositions herein are used to modulate ortreat neoplastic growth. Examples of neoplastic growth include, but arenot limited to, breast cancer, skin cancer, bone cancer, prostatecancer, liver cancer, lung cancer, brain cancer, cancer of the larynx,gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal, neuraltissue, head and neck, colon, stomach, bronchi, kidneys, basal cellcarcinoma, squamous cell carcinoma of both ulcerating and papillarytype, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma,reticulum cell sarcoma, myeloma, giant cell tumor, small-cell lungtumor, gallstones, islet cell tumor, primary brain tumor, acute andchronic lymphocytic and granulocytic tumors, hairy-cell leukemia,adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosalneuronms, intestinal ganglioneuromas, hyperplastic corneal nerve tumor,marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian tumor,leiomyomater tumor, cervical dysplasia and in situ carcinoma,neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid,topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi'ssarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renalcell tumor, polycythermia vera, adenocarcinoma, glioblastoma multiforme,leukemias, lymphomas, malignant melanomas, epidermoid carcinomas, andother carcinomas and sarcomas.

Thus, in some embodiments, a composition herein (e.g., SEQ ID NO: 1) canbe administered simultaneously with, co-formulated with, or administeredin the same therapy as an anti-neoplastic agent.

In some embodiments, the compositions herein are used to modulate andtreat abnormal temperature associated with non-rapid eye movement (NREM)during sleep, thermotaxis of human spermatozoa toward fertilization site(isthmic-ampullary junction) at ovulation, and hot flashes inpostmenopausal women.

In some embodiments, the compositions herein are used to treat orprevent plants/crops from yield losses. Examples of plants that may betreated with the compositions herein include major crops (corn,soybeans, hay, wheat, cotton, sorghum, rice, etc.) Examples ofconditions resulting in crop losses are diseases caused by bacteria,viruses, and fungi. Other examples of conditions that may result in croplosses that can be preventable or diminished by the compositions hereininclude stress conditions such as drought, freezing, oxidative stress,unfavorable or reduced temperatures, infection by pathogens and otherunfavorable environmental conditions.

In some embodiments, the compositions herein are used to modulate (e.g.,increase, decrease or control) mitochondrial activity in plants.Examples of plants that may be treated with the compositions hereininclude ornamental crops: flower bulbs (e.g., Tulips, Daffodils,Hyacinths, Crocus, Dutch iris, Allium etc.), cut flowers (e.g., roses,carnation, lily, gladiolus, bird of paradise, etc); vegetable crop(e.g., tomato, cucumber, celery, eggplants, pumpkins, carrot, lettuce,zucchini, etc.); fruit crops (e.g., apple, citrus, peach, pear, plums,banana, pineapple, olive, avocado, papaya, mango, nuts, berries, andother types of agricultural crops such as grain (e.g., corn, soybeans,hay, wheat, barley, corn, cotton, sorghum, and rice) and trees used forlumber (e.g, Douglas fir, cedar, maple, oak, poplar).

In some embodiments, the compositions herein are used to modulate (e.g.,increase, decrease or control) seed production that is regulated byplant temperature. Examples of plants that may be treated with thecompositions herein include seeds of ornamental crops, vegetable crops,fruit and nut crops, seeds of other types of agricultural crops, orother plants disclosed herein.

In some embodiments, the compositions herein are used to modulate (e.g.,increase, decrease or control) secretory products in plants or animalsthat are associated with changes in temperature. Such secretary productsinclude, but are not limited to, small volatiles and non-volatilecompounds such as terpenes, fatty acid oxidative products, and amines,as well as high molecular weight molecules such as polypeptides andpolysaccharides. Such secretions can be, for example, involved ininter-, intra-cellular communications and/or diseases.

The invention herein also provides for nucleic acids that encode thecompositions herein and nucleic acid that are complementary to nucleicacids that encode the compositions herein. Nucleic acids that encode thecompositions herein can be inserted into a vector to express thepolypeptides herein recombinantly. Nucleic acids that are complementaryto the polypeptides herein can be used as diagnostics or research toolsor to modulate the expression of certain polypeptides.

The compositions herein can be formulated with one or more carriers orexcipients for delivery to an organism, such as an animal or a plant.Such carriers can be, for example, pharmaceutical carriers, veterinarycarriers, and agricultural carriers. For delivery to an animal, thecompositions herein may be administered in a therapeutically effectivedose to reduce, inhibit, eliminate, ameliorate or prevent a condition.Similarly, for delivery to a plant (e.g., a crop plant), thecompositions herein can be delivered in an effective dose to reduce,inhibit, eliminate, ameliorate or prevent a condition.

The invention also provides for antibodies or antibody fragments thatare specific to the polypeptides herein. Such antibodies or antibodyfragments can be used therapeutically, prophylactically, or for researchpurposes. Such antibodies or antibody fragments are preferably humanizedand/or monoclonal.

The invention herein also provides for methods for screening for bindingpolypeptides (receptors) and for agents that modulate the compositionherein, or their analogs (ligands) binding to the receptors. Bindingaffinity is determined by a competitive assay using labeled agents(e.g., biotinylated or fluorescent) incubated with the receptors in thepresence of various concentrations of a composition of the invention.The affinity binding constant, K_(a), has to be of greater than or equalto about 10⁵ to 10⁷ M⁻¹, preferably of greater than or equal to about10⁸ M⁻¹, more preferably of greater than or equal to about 10⁹ M⁻¹ andstill more preferably of greater than or equal to about 10¹⁰ M⁻. Incertain embodiments binding affinity constants of peptides for thebinding polypeptides may exceed 10¹¹ to 10¹² M⁻¹. Affinities of bindingpolypeptides for ligands according to the present invention can bereadily determined using conventional techniques, for example thosedescribed by Scatchard et al. (1949 Ann. N.Y. Acad. Sci. 51:660), or byother various techniques described in the scientific literature.

The invention herein also provides for methods for preparing apeptidomimetic of the polypeptides herein.

SUMMARY OF THE FIGURES

FIG. 1 illustrates the efficacy of SEQ ID NO: 1 in relieving pain inrats 3 days after surgery.

FIG. 2 illustrates the efficacy of SEQ ID NO: 1 in relieving pain inrats 3 h after surgery

FIG. 3 illustrates the efficacy of SEQ ID NO: 1 in relieving pain inrats after surgery

FIG. 4 illustrates heat production by Sauromatum guttatum appendixtreated with aspirin (ASA) and various opioid peptides and theneurotoxic peptide β-amyloid peptide (Aβ 1-42).

FIG. 5 illustrates heat production by Sauromatum guttatum appendixtreated with salicylic acid (SA) in the presence of human opioidpeptides (β-Endorphin and Neuropeptide AF) and, β-amyloid peptide, (Aβ1-42); and a plant virulent bacterial pathogen (Pst DC3000).

FIG. 6 illustrates heat production by Sauromatum guttatum appendixtreated with 2,6-dihydroxybenzoic acid (2,6-DHBA) in the presence ofβ-amyloid peptide (Aβ 1-42), SEQ ID NO: 2, and SEQ ID NO: 1.

DETAILED DESCRIPTION OF INVENTION

The present invention relates to compositions and methods for modulatingmitochondria and mitochondrial related or metabolic related conditions.

In one aspect, a composition herein includes a polypeptide comprising,consisting essentially of, or consisting of a polypeptide selected fromthe group consisting of: SEQ ID NOs: 1-256, or more preferably selectedfrom the group consisting of: SEQ ID NOs: 1-24, 50-244, and 248-249; ormore preferably selected from the group consisting of: SEQ ID NOs: 1-2,or more preferably SEQ ID NO: 1.

In one aspect, a composition comprises a nucleic acid sequence encodingone or more of the above.

In one aspect, a composition comprises an antibody that specificallybinds an epitope comprising one or more of the above polypeptides.

In one aspect, the present invention relates to a method for identifyingnovel compositions (e.g., polypeptides, peptide nucleic acids, nucleicacids, and small molecules) that modulate the mitochondria. Such methodsinclude administering a test agent to a thermogenic plant; measuringtemperature of said thermogenic plant; and determining if said testagent modulates temperature in said plant.

In one aspect, the compositions herein are used to treat a mitochondrialor metabolic condition selected from the group consisting of: innateimmune response activation and ability to fight parasites and pathogens,pain, inflammation, temperature regulation, neoplastic growth (e.g.,cancer), skin and dermatological conditions, and neurological andneurodegenerative conditions.

DEFINITIONS

The term “agonist” as used herein refers to a compound, molecule, oragent that stimulates a biological activity. Examples of agonistmolecules include, but are not limited to, agonists that stimulatereceptors, e.g., morphine antagonist of the opiate μ receptors.

The term “amino acid” or “amino acid residue” refers to an amino acid,which is preferably in the L-isomeric form. When an amino acid residueis part of a polypeptide chain, the D-isomeric form of the amino acidcan be substituted for the L-amino acid residue, as long as the desiredfunctional property is retained. NH₂ refers to the free amino grouppresent at the amino terminus of a polypeptide. COOH refers to the freecarboxyl group present at the carboxyl terminus of a polypeptide. Theamino acids herein can be represented by their standard 1-letter code or3-letter code. An amino acid residue represented by “X” or “Xxx” refersto any one of the naturally occurring or non-naturally occurring aminoacid residues known in the art or to a modification of a nearby residue.In keeping with standard protein nomenclature described in J. Biol.Chem., 1969, 247:3552-59, and adopted at 37 C.F.R. §§1.821-2461.822, allamino acid residue sequences represented herein by formulae have a leftto right orientation in the conventional direction of amino-terminus tocarboxyl-terminus. In addition, the phrase “amino acid residue” isbroadly defined to include modified and unusual amino acids, such asthose referred to in 37 C.F.R. §§1.821-1.822, and incorporated herein byreference. In a peptide or polypeptide, suitable conservativesubstitutions of amino acids are known to those of skill in this art andcan be made generally without altering the biological activity of theresulting molecule. Watson et al., book (1987, Molecular Biology of theGene, 4th Edition, The Benjamin Cummings Pub. Co., p. 224), isincorporated herein by references. Amino acid substitutions aretypically of single residues, such substitutions are preferably madewith those set forth in Table I., but may be of multiple residues,either clustered or dispersed. An amino acid can be replaced with adifferent naturally occurring or a non-conventional amino acid residue.Such substitutions may be classified as “conservative”, in which case anamino acid residue contained in a polypeptide is replaced with anothernaturally occurring amino acid of similar character either in relationto polarity, side chain functionality or size. Additions encompass theaddition of one or more naturally occurring or non-conventional aminoacid residues. Deletion encompasses the deletion of one or more aminoacid residues.

TABLE I Conservative amino acid substitution Original residueConservative substitution(s) Ala Gly; Ser Arg Lys Asn Gln; His Cys SerGln Asn Glu Asp Gly Ala; Pro His Asn; Gln Ile Leu; Val Leu Ile; Val LysArg; Gln; Glu Met Leu; Tyr, Ile Phe Met; Leu; Tyr Ser Thr Thr Ser TrpTyr Tyr Trp; Phe Val Ile; Leu

Substitutions encompassed by the present invention may also be“non-conservative”, in which an amino acid residue which is present in apeptide is substituted with an amino acid having different properties,such as naturally-occurring amino acid from a different group (e.g.,substituting a charged or hydrophobic amino acid with alanine), oralternatively, in which a naturally-occurring amino acid is substitutedwith a non-conventional amino acid.

The term “analog(s)” as used herein refers to a composition that retainsthe same structure or function (e.g., binding to a receptor) as apolypeptide or nucleic acid herein, such as the same gene from adifferent organism. Examples of analogs include mimetics orpeptidomimetics, peptide, nucleic acids, small and large organic orinorganic compounds, as well as derivatives and variants of apolypeptide or nucleic acid herein. Such derivatives and variants referto peptides and nucleic acids that differ from the naturally occurringpolypeptides and nucleic acids by one or more amino acid or nucleic aciddeletions, additions, substitutions or side-chain modifications. In someembodiments, a peptide analog is a peptide in which one or more of theamino acids has undergone side-chain modifications. Examples ofside-chain modifications contemplated by the present invention includemodifications of amino groups such as by reductive alkylation byreaction with an aldehyde followed by reduction with NaBH₄; amidinationwith methylacetimidate; acylation with acetic anhydride; carbamoylationof amino groups with cyanate; trinitrobenzylation of amino groups with2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groupswith succinic anhydride and tetrahydrophthalic anhydride; andpyridoxylation of lysine with pyridoxal-5-phosphate followed byreduction with NaBH₄. In some embodiments, a peptide analog is one inwhich the guanidine group of arginine residue(s) is modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal; carboxyl group(s) ismodified by carbodiimide activation via O-acylisourea formation followedby subsequent derivitisation, for example, to a corresponding amide;sulphydryl group(s) may be modified by methods such ascarboxymethylation with iodoacetic acid or iodoacetamide; performic acidoxidation to cysteic acid; formation of a mixed disulphides with otherthiol compounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH. In any of theanalogs herein, any modification of cysteine residues preferably do notaffect the ability of the peptide to form the necessary disulphidebonds. In some embodiments, a peptide analog comprises tryptophanresidue(s) that are modified by, for example, by oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides; tyrosine residuesaltered by nitration with tetranitromethane to form a 3-nitrotyrosinederivative; imidazole ring(s) of a histidine residue modificationaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate; proline residue(s)modified by, for example, hydroxylation in the 4-position; glycosylationvariants from a completely unglycosylated molecule to a modifiedglycosylated molecule; and altered glycosylation patterns as a resultfrom expression of recombinant molecules in different host cells.

The term “antagonist” as used herein refers to a compound, molecule, oragent that inhibits a biological activity. Examples of antagonistmolecules include, but are not limited to, peptides, small molecules,antibodies, antisense nucleic acids, siRNA nucleic acids, and otherbinding agents.

The term “antibody” is used in the broadest sense and specificallycovers, for example, polyclonal antibodies, monoclonal antibodies (mAbs)(including agonist, antagonist, and neutralizing antibodies), chimericantibodies, antibody compositions with mono and polyepitopicspecificity, single chain antibodies, anti-idiotypic (anti-Id)antibodies to antibodies that can be labeled in soluble or bound form,polymers and conjugates of immunoglobulins, as well as fragments,regions or derivatives thereof (e.g., separate heavy chains, lightchains, Fab, F(ab′)₂, Fabc, and Fv). Antibody fragments can be preparedfor example by enzymatic cleavage of antibodies with enzymes such aspepsin or papain. Antibody aggregates, polymers and conjugates can begenerated by diverse methods, e.g. by thermal treatment, reaction withsubstances such as glutaraldehyde, reaction with immunoglobulin-bindingmolecules, biotinylation of antibodies and subsequent reaction withstreptavidin or avidin. The term “monoclonal antibody” as used hereinrefers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts.

The term “antigens” includes monovalent and polyvalent antigens. Apolyvalent antigen is a molecule or a molecule complex to whichsimultaneous binding of more than one immunoglobulin is possible,whereas a monovalent antigen can bind only a single antibody at eachparticular time. Hapten is normally the designation given to a moleculewhich is not immunogenic per se but which is normally bound to a carrierfor immunization purposes.

The term “effective amount” as used herein when referring to acomposition means the amount or dosage of that composition that isrequired to induce a desired effect. In some embodiments, an effectivedose refers to an amount that is required to induce a local analgesic,anti-pyrogenic, flowering, pesticide, anti-dementia, and/oranti-inflammatory effect.

The term “fragment” as used herein refers to a portion of a composition.For example, when referring to a polypeptide, a fragment of apolypeptide is some but not the entire amino acid polymer that comprisesthe polypeptide. A polypeptide fragment can have up to 99, 95, 90, 85,80, 75, 70, 65, or 60% of the sequence of the parent polypeptide. Insome embodiments, a fragment has between 3-40, 3-30, 4-20, or 4-10 aminoacids of the parent sequence.

The terms “gene therapy” and “genetic therapy” refer to the transfer ofheterologous nucleic acids to the certain cells, target cells, of amammal, particularly a human, with a disorder or conditions for whichsuch therapy is sought. The nucleic acid is introduced into the selectedtarget cells in a manner such that the heterologous DNA is expressed anda therapeutic product encoded thereby is produced. Alternatively, theheterologous nucleic acids can in some manner mediate expression of anucleic acid that encodes the therapeutic product; it can encode aproduct such as a peptide or RNA that in some manner mediates, directlyor indirectly, expression of a therapeutic product. Genetic therapy canalso be used to nucleic acid encoding a gene product replace a defectivegene or supplement a gene product produced by the mammal or the cell inwhich it is introduced.

The term “homolog” when referring to a polymer (e.g., a peptide or anucleic acid) refers to a second polymer that has at least about 50sequence identity, more preferably at least 55% sequence identity, morepreferably at least 60% sequence identity, more preferably at least 65%sequence identity, more preferably at least 70% sequence identity, morepreferably at least 55% sequence identity, more preferably at least 80%sequence identity; or preferably at least about 81% sequence identity,more preferably at least about 82% sequence identity, yet morepreferably at least about 83% sequence identity, yet more preferably atleast about 84% sequence identity, yet more preferably at least about85% sequence identity, yet more preferably at least about 86% sequenceidentity, yet more preferably at least about 87% sequence identity, yetmore preferably at least about 88% sequence identity, yet morepreferably at least about 89% sequence identity, yet more preferably atleast about 90% sequence identity, yet more preferably at least about91% sequence identity, yet more preferably at least about 92% sequenceidentity, yet more preferably at least about 93% sequence identity, yetmore preferably at least about 94% sequence identity, yet morepreferably at least about 95% sequence identity, yet more preferably atleast about 96% sequence identity, yet more preferably at least about97% sequence identity, yet more preferably at least about 98% sequenceidentity and yet more preferably at least about 99% sequence identityand preferably the same function. For example, a polypeptide homologousto any of the polypeptides herein (e.g., SEQ ID NOs: 1-256) is one thatpreferably has at least 80% sequence identity and similar function ofmodulating mitochondrial activity, or more preferably acting as anagonist or antagonist to SEQ ID NO: 5 or agonist to heat production of2,6-DHBA.

The term “isolated” means altered from its natural state; i.e., if itoccurs in nature, it has been changed or removed from its originalenvironment, or both. For example, a naturally occurring polynucleotideor a polypeptide naturally present in a living animal in its naturalstate is not “isolated”, but the same polynucleotide or polypeptideseparated from the coexisting materials of its natural state is“isolated”, as the term is employed herein. For example, with respect topolynucleotides, the term isolated means that it is separated from thenucleic acid and cell in which it naturally occurs.

The term “protein”, “peptide”, “oligopeptides” or “polypeptide” as usedherein refers to any composition that includes two or more amino acidsjoined together by a peptide bond. It will be appreciated thatpolypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids, and thatmany amino acids, including the terminal amino acids, may be modified ina given polypeptide, either by natural processes such as glycosylationand other post-translational modifications, or by chemical modificationtechniques which are well known in the art. Among the knownmodifications which may be present in polypeptides of the presentinvention include, but are not limited to, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of a flavanoid or aheme moiety, covalent attachment of a polynucleotide or polynucleotidederivative, covalent attachment of a lipid or lipid derivative, covalentattachment of phosphatidylinositol, cross-linking, cyclization,disulfide bond formation, demethylation, formation of covalentcross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycation, glycosylation,glycosylphosphatidyl inositol (GPI) membrane anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto polypeptides such as arginylation, and ubiquitination. The term“protein” also includes “artificial proteins” which refers to linear ornon linear polypeptides, consisting of alternating repeats of a peptide(e.g., SEQ ID NO: 1-256), and a spacer. A DNA-construct encoding thepeptide and a spacer alternate repeats can be synthesized using methodsknown in the art. (Rötzschke et al., 1997, Proc. Natl. Acad. Sci. USA94:14642-14647). The above methods allow for the amplification of theantigenicity of the peptide and for insertion into an expression vectorat high levels.

The term “opioid” as used herein means all agonists and antagonists ofopioid receptors, such as mu (μ), kappa (κ), and delta (δ) opioidreceptors and subtypes thereof. For a discussion of opioid receptors andsubtypes see Goodman and Gilman's The Pharmacological Basis ofTherapeutics 9th ed. J. G. Harman and L. E. Limird Eds., McGraw-Hill NewYork, 1996, pp. 521-555, which is incorporated herein by reference forall purposes. The opioid can be any opioid receptor agonist orantagonist known or to be developed. Preferred opioids interact with theμ-opioid receptor or the κ- and δ-opioid receptors. Preferably, theopioid is an opioid-receptor agonist.

The term “organism” as used herein can be, for example, a microorganism(e.g., virus or bacteria), plant (e.g., crop plants such as soy, wheat,barley, rice, corn, sugar, etc.), or animal. Animals include bothmammals (e.g., farm animals, donkeys, goats, chicken, rabbits, dogs,cats, swine, cattle, sheep, horses, or primates) and non-mammals, e.g.,(e.g., insects and birds). Preferably an animal is a mammal, or morepreferably a human.

The term “purified” as used herein to describe a polypeptide,polynucleotide, or other compositions, refers to such polypeptide,polynucleotide, or other composition separated from one or morecompounds which are usually associated with it in nature. Such othercompositions can be, for example, other polypeptides or polynucleotides,carbohydrates, lipids, etc. The term “purified” can also be used tospecify the separation of monomeric polypeptides of the invention fromoligomeric forms such as homo- or hetero-dimers, trimers, etc. The term“purified” may also be used to specify the separation of covalentlyclosed (i.e. circular) polynucleotides from linear polynucleotides. Asubstantially pure polypeptide or polynucleotide typically comprises atleast about 50%, 60%, 70%, 80%, or 90% weight/weight of a polypeptide orpolynucleotide sample, or more preferably at least about 95%, 96%, 97%,98%, 99%, or 99.5% weight/weight of a polypeptide or polynucleotidesample. As a preferred embodiment, a polypeptide or polynucleotide ofthe present invention is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 96%, 96%, 98%, 99%, or 100% pure relative to heterologouspolypeptides and polynucleotides, respectively.

Compositions

The compositions herein are based on a class of phylogenetically relatedplant reductases: pinoresinol-lariciresinol reductase, isoflavonereductase, and phenylcoumaran benzylic ether reductase (Gang et al., J.Biol. Chem., 1999, 274:7516-7527) having sequences SEQ ID NOs: 245-247,human beta-neoendorphin-dynorphin precursor (Proenkephalin B) havingsequence SEQ ID NO: 250; protein from Arabidopsis thaliana (E71436)having sequence SEQ ID NO: 253, Epsin 1, EPS-15 interacting protein 1derived from human having sequence SEQ ID NO: 254, phosphatidylinositolpolyphosphate 5-phosphatase isoform derived from human having sequenceSEQ ID NO: 255, and RNA2 polyprotein (P2) derived from ssRNApositive-strand viruses, having sequence SEQ ID NO: 256.

SEQ ID NO: 245 Arabidopsis phenylcoumaran benzylic ether reductasehomolog Tp1 [Thuj plicata (ACCESSION AAF64183) having sequence

MDKKSRVLIVGGTGFIGKRIVKASLALGHPTYVLFRPEALSYIDKVQMLISFKQLGAKLLEASLDDHQGLVDVVKQVDVVISAVSGGLVRHHILDQLKLV EAIKEAGNIKR

EFGMDPDVVEDPLEPGNITFIDKRKVRRAIEAATIPYTYVSSNMFAGFFAGSLAQLQDAPRMMPARDKVLIYGDGNVKGVYVDEDDAGIYIVKSIDDPRTLNKTVYIRPPMNILSQKEVVEIWERLSGLSLEKIYVSEDQLLNMKDKSYVEKMARCHLYHFFIKGDLYNFEIGPNATEGTKLYPE VKYTTMDSYMERYL

SEQ ID NO 246 is pinoresinol-lariciresinol reductase from Arabidopsisthaliana (ACCESSION NM_(—)102944) having sequence

MGESKRTEKTRVLVVGATGYIGKRIVRACLAEGHETYVLQRPEIGLEIEKVQLFLSFKKLGARIVEGSFSDHQSLVSAVKLVDVVVSAMSGVHFRSHNIL VQLKLVEAIKEAGNVKR

EFGMDPPRMGHALPPGRETFDQKMERQAIEAAGIPYTYVVGACFAAYFAGNLSQMVTLLPPKEKVNIYGDGNVKVVFADEDDIAKYTAKTLNDPRTLNKTVNIRPPDNVLTQLELVQIWEKLTGKELEKTNIAAQDFLANIEQMEIPHQAGIGHFYHIFYEGCLTDHEVGEDEEASSLY PDVKYKRMDDYLRMFL

SEQ ID NO: 247 is Arabidopsis thaliana mRNA for isoflavonoid reductasehomologue (ACCESSION Z49777) having sequence

MATEKSKILVIGGTGYIGKFLVEASAKAGHSTFALVREATLSDPVKGKTVQSFKDLGVTILHGDLNDHESLVKAIKQVDVVISTVGSMQILDQTKIISAI KEAGNVKR

EFGVDVDRTSAVEPAKSAFAGKIQIRRTIEAEGIPYTYAVTGCFGGYYLPTLVQFEPGLTSPPRDKVTILGDGNAKAVINKEEDIAAYTIKAVDDPRTLNKILYIKPSNNTLSMNEIVTLWEKKIGKSLEKTHLPEEQLLKSIQESPIPINVVLSINHAVFVNGDTNISIEPSFGVEASELYPDVKYT SVDEYLSYFA

SEQ ID NO: 250 is human beta-neoendorphin-dynorphin precursor(Proenkephalin B) (Preprodynorphin), (ACCESSION P01213) having sequence

MAWQGLVLAACLLMFPSTTADCLSRCSLCAVKTQDGPKPINPLICSLQCQAALLPSEEWERCQSFSFFTPSTLGLNDKEDLGSKSVGEGPYSELAKLSGS FLKELEKSK

ISTKENTLSKSLEEKLRGLSDGFREGAESELMRDAQLNDGAMETGTLYLAEEDPKEQVKRYGGFLRKYPKRSSEVAGEGDGDSMGHEDLYKRYGGFLRRIRPKLKWDNQKRYGGFLRRQFKVVTRSQEDPNAYSGEL FDA

SEQ ID NO: 253 is a protein from Arabidopsis thaliana (E71436) havingsequence:

MVKKIANDVSNKLFPLPKGFGDFVGIEDHIKAIKSILCLESKEARIMVGIWGQSGIGKSTIGRALFSQLSSQFHHRAFITYKSTSGSDVSGMKLSWEKELLSEILGQKDIKIDHFGVVEQRLKHKKVLILLDDVDNLEFLKTLVGKAEWFGSGSRIIVITQDKQLLKAHEIDLVYEVELPSQGLALKMISQYAFGKDSPPDDFKELAFEVAELVGSLPLGLSVLGSSLKGRDKDEWVKMMPRLRNDSDDKIEETLRVGYDRLNKKNRDNVKELLEDDVGLTMLADKSLIRITPDGDIEMHNLLEKLGREIDRAKSKGNPAKRQFLTNFEDIQEVVTEKTGTETVLGIRVPPTVLFSTRPLLVINEESFKGMQIGLWSKIDLPQGLVYLPLKLKLLKWNYCPLKSLPSTFKAEYLVNLIMKYSKLEKLWEGTLPLGSLKKMDLGCSNNLKEIPDLSLAINLEELNLSKCESLVTLPSSIQNAIKLRTLYCSGVLLIDLKSLEGMCNLEYLSVDWSSMEGTQGLIYLPRKLKRLWWDYCPVKRLPSNFKAEYLVELRMENSDLEKLWDGTQPLGSLKEMYLHGSKYLKEIPDLSLAINLERLYLFGCESLVTLPSSIQNATKLINLDMRDCKKLESFPTDLNLESLEYLNLTGCPNLRNFPAIKMGCSYFEILQDRNEIEVEDCFWNKNLPAGLDYLDCLMRCMPCEFRPEYLTFLDVSGCKHEKLWEGIQIHALLDGYELAGHLDGSIETPAPTLTTNNVVSANPQYTLWKRQDRLIFSALIGAISPPVQPLVSRATKASQIWKTLTNTYAKSSYDHIKQLRTQIKQLKKGTKTIDEYVLSHTTLLDQLAILGKPMEHEEQVERILEGLPEDYKTVVDQIEGKDNTPSITEIHERLINHEAKLLSTAALSSSSLPMSANVAQQRHHNNNRNNNQNKNRTQGNTYTNNWQPSANNKSGQRPFKPYLGKCQICNVQGHSARRCPQLQAMQPSSSSSASTFTPWQPRANLAMGAPYTANNWLLDSGATHHITSDLNALALHQPYNGDDVMIADG TSLKITKTGST

NARDLTLNKVLYVPDIQKNLVSVYRLCNTNQVSVEFFPASFQVKDLNTGTLLLQGRTKDELYEWPVTNPKATALFTTPSPKTTLSSWHSRLGHPSSSILNTLISKFSLPVSVSASNKLACSDCFINKSHKLPFSISSIKSTSPLEYIFSDVWMSPILSPDNYKYYLQKSQVKSTFIAFKALVENRFQAKIRTLYSDNGGEFIALREFLVSNGISHLTSPPHTPEHNGLSERKHRHIVETGLTLLTQASVPREYWPYAFAAAVYLINRMPTPVLSMESPFQKLFGSKPNYERLRVFGCLCFPWLRPYTHNKLEERSRRCVFLGYSTQTAYLCFDVEHKRLYTSRHVVFDEASFPFSNLTSQNSLPTVTFEQSSSPLVTPILSSSSVLPSCLSSPCTVLHQQQPPVTTPNSPHSSQPTTSPAPLSPHRSTTMDFQVPQPTAPNENGPEPEAQSPPIGPLSNPTHEAFIGPLPNPNRNPTNEIEPTPAPHPKPVKPTTTTTTPNRTTVSDASHQPTAPQQNQHNMKTRAKNNIKKPNTKFSLTATLPNRSPSEPTNVTQALKDKKWRFAMSDEFDAQQRNHTWDLVPHESQLLVGCKWVFKLKYLPNGAIDKYKARLVAKGFNQQYGVDYAETFSPVIKSTTIRLVLDVAVKKDWEIKQLDVNNAFLQGTLTEEVYMAQPPGFIDKDRPTHVCRLRKAIYGLKQAPRAWYMELKQHLFNIGFVNSLSDASLFIYWSDKSSIDAVLTSLAERFSIKDPTDLHYFLGIEATRTKQGLHLMQRKYIKDLLAKHNMADAKPVLTPLPTSPKLTLHGGTKLNDASEYRSVVGSLQYLAFTRPDIAYAVNRLSQLMPQPTEDHWQAAKRVLRYLAGTSTHDWAGDSDDYVSTNAYVIYLGKNPISWSSKKQRGVARSSTESEYRAVANAASEVKWLCSLLSKLHIRLPIRPSIFCDNIGATYLCANPVFHSRMKHIAIDYHFVRNMIQSGALRVSHVSTRDQLADALTKPLSRAHFQSARFKIGVRQLPPS

SEQ ID NO: 254 is Epsin 1, EPS-15 interacting protein 1 derived fromhuman (Accession No. O88339) having sequence:

MSTSSLRRQMKNIVHNYSEAEIKVREATSNDPWGPSSSLMSEIADLTYNVVAFSEIMSMIWKRLNDHGKNWRHVYKAMTLMEYLIKTGSERVSQQCKENMYAVQTLKDFQYVDRDGKDQGVNVREKAKQLVALLRDEDRLREERAHALKTKEKLAQTATASSAAVGSGPPPEAEQAWPQSSGEEELQLQLALAMSKEEADQPPSCGPEDDVQLQLALSLSREEHDKEERIRRGDDLRLQMAIEESKRETGGKEESSLMDLADVFTTPAPPQASDPWGGPASVPTAVPVAAAASDPWGAPAVPPAADPWGGAAPTPASGDPWRPAAPTGPSVDPWGGTPAPAAGEGPTSDPWGSADGGAPVSGPPSSDPWAPAPAFSDPWGGSPAKPSSNGTAVGGFDTEPDEFSDFDRLRTALPTSGSSTGELELLAGEVPARSPGAFDMSGVGGSLAESVGSPPPAATPTPTPPTRKTPESFLGPNAALVDLDSLVSRPGPTPPGAKAS NP

GAPATGPSVTNPFQPAPPATLTLNQLRLSPVPPVPGAPPTYISP LGGGPGLPPM MPPGPPAPNT NPFLL

SEQ ID NO: 255 is phosphatidylinositol polyphosphate 5-phosphataseisoform derived from human (Accession No. NP_(—)000267) having sequence:

MEPPLPVGAQPLATVEGMEMKGPLREPCALTLAQRNGQYELIIQLHEKEQHVQDIIPINSHFRCVQEAEETLLIDIASNSGCKIRVQGDWIRERRFEIPDEEHCLKFLSAVLAAQKAQSQLLVPEQKDSSSWYQKLDTKDKPSVFSGLLGFEDNFSSMNLDKKINSQNQPTGIHREPPPPPFSVNKMLPREKEASNKEQPKVTNTMRKLFVPNTQSGQREGLIKHILAKREKEYVNIQTFRFFVGTWNVNGQSPDSGLEPWLNCDPNPPDIYCIGFQELDLSTEAFFYFESVKEQEWSMAVERGLHSKAKYKKVQLVRLVGMMLLIFARKDQCRYIRDIATETVGTGIMGKMGNKGGVAVRFVFHNTTFCIVNSHLAAHVEDFERRNQDYKDICARMSFVVPNQTLPQLNIMKHEVVIWGDLNYRLCMPDANEVKSLINKKDLQRLLKFDQLNIQRTQKKAFVDFNEGEIKFIPTYKYDSKTDRWDSSGKCRVPAWCDRILWRGTNVNQLNYRSHMELKTSDHKPVSALFHIGVKVVDERRYRKVFEDSV RIMDRMEND

LELSRREFVFENVKFRQLQKEKFQISNNGQVPCHFSFIPKLNDSQYCKPWLRAEPFEGYLEPNETVDISLDVYVSKDSVTILNSGEDKIEDILVLHLDRGKDYFLTISGNYLPSCFGTSLEALCRMKRPIREVPVTKLIDLEEDSFLEKEKSLLQMVPLDEGASERFLQVPKEIWLLVDHLFKYACHQEDLFQTPGMQEELQQIIDCLDTSIPETIPGSNHSVAEALLIFLEALPEPVICYELYQRCLDSAYDPRICRQVISQLPRCHRNVFRYLMAFLRELLKFSEYNSVNANMIATLFTSLLLRPPPNLMARQTPSDRQRAIQF LLGFLLGSEED

SEQ ID NO: 256 is RNA2 polyprotein (P2) derived from ssRNApositive-strand viruses, Comoviridae (Accession No. Q9YK98) havingsequence:

MSESGNTTSMPGCGRMCALRSTWSKRAFLVACKDGALTSDGRCPQYGCGALVSITKGVQQPKKTASAKVVKCLCWVQPARWCEKHSKGPASPNGSVTTKRSNSARAAPAPLPYKKQTCDVVVTVGPLELVYPALVSEELPTPVAATPTKVEEVPIPELPLWLAPAWMVEQPYAATPEVLCLTQREEFALLKKRLTRKGKLLQRRATHARFEARAALARVRAATQRKVEEVTALVIKGRRILAAHQLLRELEEVAPLSQAQEQLVASSCAAAAARQEECASFLRRAKAWRKSISATPPVAATAVASKVVSATMPWAHLGLSLGGLLAVPTLDGTLGAKQWNAKTIATWVLKPVVSCVQSVHAKVRDWLHSQPEVGVTNTKVPLVLPEVCLGVLSPPSLSEEIVDNPQETSQSGIWHPEMGVRNIYVFHDDSWETSPEEDENYTYTFSRQCGIPYLLVEGRGAEERKNTILGWDFSLHNDGFE

PEEGYTKELVTPVAL EEEDKYSTASSCGFFSLDDVSSAITIQCPGLLSADADVHFFDGPGYRCSSRPRDFRPPVVRGCDYESRVKASIQRKIENPLQERFITVLREKRKKNKKKEFHSFSACFAFKRKQIQWPPTPNEMVNEWEEYCIAQAWLPFEVVVTDEIEDVTPLYPGGRDYNCNSQLLFPLAPLSTVYCDDSCFHPNDGWTTDGNGKHFRLSPQFVLPDVPIPIVHRVTRQLPQFLYDLGIGDLTCNSGYQAENLQEEIQERMEDRSEEKPVPSLDTLISKLSKRSTKVKGAGENRYADRHSLTEKAIFHQPGALSRMRSGKEKTIVAANHNSDQISVRMAECGKPVFTPLPRMSDEMLRKFLEKGLGSTSTVALDIGIQSHIPQGMPTVAFVNVMDTRIEDPLYSSLCGSYIDLGRDRAKTLCLPLVNFPMSKLAEDVDDVLNGLMLCTHFQDSTKFGVGKPAFQYGTLEFQEFKPSAYSDFSRVRDNWDAIAKQQNTPNDRILAGFSVLGAVSQAYNQALPVFKSVELVAPPKRKPVVATFQNPTTLGRSNTTRSFRMPTMDLPRSTGRDAPIPIVHRRNNNDVHFDEATPARFSTCDSGLVADTTLAFAKMYQCKKDAKAGHVLATIDIQECVFEDNRRVALDWLAHGLASFKYDLQLTVDSNPFVGVTLGITVDAFDRLLPQISDEVIAVPLAFQLPTYLFPISKKGTFTQTIDFAAIAGYNFFPHVAAFGRPKIIVYIVSDNDLPASDTWMCLVELHMTRLESSTLACSPTLVLPQAFGGDLPLDLWRGPYTFPLGGGTKRLSTSLDIGTSTTTVSGWRTVSPAAYALFLQGHGGSLVGEVVHTGSAAVSCALHLCISFGGAPPTLEEALVFPGFRLPSGEGKFHIKVQTPYGRLSTLTPDCALYVYLAGGPIAVAPMSVPYQFCIHLERLVDDGAPPRTIGLIREFNWATINNFKSDDITFAIPARLSDLVLTCGDVTMSTNPLALLIGSCGFFRGNLTVVLEWATFLKAGDKEGTVQLTTCRGMINNVKGVRNAIQKKVVNLSLVGSVSRYLNVGDFTGFAQSGGQVGYDEIFLEFSTNKAKQIRYLNINVELDENFELYGRT IIPLKNTAPAFASTSSAPNES

Peptides opioids derived from SEQ ID NOs: 245-247 include those of SEQID NOs: 1-24 and 50-152. Known peptides opioids derived from SEQ ID NO:250 are β-neoendorphin (SEQ ID NO: 16); Dynorphin A (SEQ ID NO: 19);Leumorphin (SEQ ID NO: 249); Rimorphin (SEQ ID NO: 248); Leu-enkephalin(SEQ ID NO: 15). In addition, SEQ ID NO: 1 is also derived from SEQ IDNO: 250, and 253-256. These peptides are shown highlighted in thesequence listing above.

In some embodiments, the compositions herein are synthetic peptidesderived from or fragments of the above gene sequences. Such peptidefragments include those having an amino acid sequence comprising,consisting essentially of or consisting of SEQ ID NOs: 1-14, 50-152 and248-249 including homologs and analogs thereof. When a polypeptide ofthe invention comprises any of the above peptide sequences, additionalamino acid residues in its C-terminus or N-terminus can be derived fromany of the above gene sequence or any sequences that are homologs oranalogous to the above gene sequences. For example, SEQ ID NO: 1 isderived from SEQ ID NO: 250 as well as SEQ ID NO: 246. Thus, in someembodiments, the present invention contemplates a 5-mer derived from SEQID NO 250 such as KFLPS (SEQ ID NO: 257) or FLPSI (SEQ ID NO: 258) or a5-mer derived from SEQ ID NO: 246 such as RFLPS (SEQ ID NO: 259) orFLPSE (SEQ ID NO: 260). In any of the embodiments herein, a polypeptidecan be a 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer,11-mer, 12-mer, 13-mer, 14-mer, 15-mer, or larger polypeptides, but ispreferably up to 40, 30, 20, or 10 amino acids long.

To eliminate vulnerability to degradation once inside the cell, thereverse sequences of any of the polypeptides herein can be used (with orwithout D-amino acids), especially the polypeptides comprising,consisting essentially of, or consisting of: SEQ ID NOs: 1-152, and245-256. Such reverse sequences include those in SEQ ID NOs: 153-244.The reverse polypeptides can retain essentially the same function (e.g.,relieving pain) but are resistant to degradation.

In preferred embodiments, a composition comprises a polypeptideconsisting of SEQ ID NOs: 1-14, 50-152, 248-249, or more preferably SEQID NOs: 1, 2, and 15 or homologs, analogs, or fragments thereof. Thesequence of SEQ ID NOs: 1 and 2 is found in both humans and plants. Insome embodiments, the invention relates to SEQ ID NO: 3 and variousfragments of SEQ ID NO: 3, such as SEQ ID NOs: 2-14.

In some embodiments, the compositions herein include a peptide havingthe reverse amino acid sequence of any of the above amino acidsequences, such as SEQ ID NOs: 153-244.

In one aspect, the present invention contemplates the above syntheticpeptides and all other peptide fragments and derivatives frompinoresinol-lariciresinol reductase, isoflavone reductase, andphenylcoumaran benzylic ether reductase. In one embodiment, the presentinvention relates to a peptide fragment from pinoresinol-lariciresinolreductase, which is between 3-50, 3-40, 3-30, 3-20, or 3-10 amino acidsin length. In another embodiment, the present invention relates to apeptide fragment from isoflavone reductase which is between 3-50, 3-40,3-30, 3-20, or 3-10 amino acids in length. In another embodiment, thepresent invention relates to a peptide fragment from phenylcoumaranbenzylic ether reductase which is between 3-50, 3-40, 3-30, 3-20, or3-10 amino acids in length. Preferably, such peptides have at least onephenylalanine or at least two phenylalanines. In some embodiments, suchpeptides comprise the sequence Phe-Leu-Pro-Ser (SEQ ID NO: 1). Any ofthe peptides herein are contemplated in both their forward and reversesequences. SEQ ID NOs: 152-244 are the reverse sequences of SEQ ID NOs:1-14 and 50-151.

In another aspect, the present invention relates to nucleic acids thatencode any of the above peptides and antibody that specifically bind anyof the above peptides.

In some embodiments, the above compositions (e.g., peptides) can be usedto modulate effects of SEQ ID NO: 49, which is the Alzheimer's peptide,Aβ 1-42.

In some embodiments, the above compositions (e.g., peptides) can be usedmodulate or enhance the effects of SEQ ID NOs: 15-48, 248-249, which areknown opioid peptides.

In some embodiments, the above compositions (e.g., peptides) can be usedto create a library to analyze functionality of compounds andcompositions that regulate mitochondria activities.

In some embodiments, the above compositions (e.g., peptides) can be usedfor screening antagonists, agonists, and modulators of differentmitochondrial activities.

1. Polypeptides

The present invention relates to composition comprising, consistingessentially of, or consisting of one or more polypeptides that comprise,consist essentially of, or consist of an amino acid sequence selectedfrom the group of SEQ ID NOs: 1-244, 248-249, as well as the sequenceswhich are the reverse of the above and any homologs, analogs, salts,prodrugs, fragments, metabolites, and combination thereof. Preferably, acomposition herein comprises a polypeptide having an amino acid sequencecomprising SEQ ID NOs 1-14 or 50-244, 248-249, or consisting essentiallyof a polypeptide having an amino acid sequence comprising SEQ ID NOs:1-14 or 50-244, 248-249, or consisting of a polypeptide having an aminoacid sequence comprising SEQ ID NO: 1-14 or 50-244, 248-249, orcomprising SEQ ID NOs: 1-2, or consisting essentially of SEQ ID NOs:1-2, or consisting of SEQ ID NOs: 1-2, or comprising of SEQ ID NO: 1, orconsisting essentially of SEQ ID NO: 1, or consisting of SEQ ID NO: 1.

In some embodiments, a composition comprises more than 1, 2, 3, 4, and 5of the polypeptides above.

The polypeptides herein may be created synthetically by any means knownin the art (synthetically synthesized or using recombinant DNAtechnology). In some embodiments, they may include an additionalmethionine at the N-terminus (e.g., SEQ ID NO: 105, MFAGYFAG) or anN-terminus methionine may be included on to them.

In some embodiments, a polypeptide herein has up to about 100, 95, 90,85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or more preferably 4 amino acidsresidue; or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50, amino acid residues;or between about 2-50, 2-40, 2-30 or 2-10 amino acid residues. In someembodiments, a polypeptide herein has up to 500,000, 100,000, 75,000,50,000, 15,000, 14,000, 13,000, 12,000, 11,000, 10,000, 9,000, 8,000,7,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,000, 900, 800, 700, 600,500, 400, 300, or 200 Daltons. In some embodiments, a polypeptide hereinhas between 200-200,000, 300-100,000,400-50,000, or 500-1000 Daltons.

The polypeptides herein are preferably isolated such that it is free ofother compounds or molecules that it normally is associated with invivo. For example, an isolated peptide of the invention can constituteat least about 50%, more preferably about 55%, more preferably about60%, more preferably about 65%, more preferably about 70%, morepreferably about 75%, more preferably about 80%, more preferably about85%, more preferably about 90%, more preferably about 95%, or morepreferably about 99% w/w of a sample containing it. In some embodiments,a polypeptide of the invention is purified.

In some embodiments, a polypeptide herein is modified or adapted forslow-release. Such modification can include substitution of one or more,2 or more, 3 or more, or 4 or more amino acids residues from an L-aminoacid residue to a D-amino acid residue. In some embodiments at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the residuein a polypeptide are D-amino acids.

In some embodiments, a polypeptide of the present invention includes oneor more post-translational modifications, e.g., N-linked or O-linkedcarbohydrate chains, processing of N-terminal or C-terminal ends,attachments of chemical moieties to the amino acid backbone, chemicalmodifications of N-linked or O-linked carbohydrate chains, and additionor deletion of an N-terminal methionine residue as a result ofprokaryotic host cell expression. The polypeptides may also be modifiedwith a detectable label, such as an enzymatic, fluorescent, isotopic oraffinity label to allow for detection and isolation of the polypeptide.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention, which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity. See U.S. Pat. No.4,179,337. The chemical moieties for derivitization may be selected.See, U.S. Pat. No. 4,179,337, which is hereby incorporated by referencein its entirety. The chemical moieties for derivitization may beselected from water-soluble polymers such as polyethylene glycol (PEG),copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. Such derivitization may occur at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

For example, in some embodiments the chemical moiety used forderivitization may be a polymer of any molecular weight, and may bebranched or unbranched. If PEG is used for derivitization, the preferredmolecular weight is between about 1 kDa and about 100 kDa (the term“about” indicating that in preparations of PEG, some molecules willweigh more, some less, than the stated molecular weight) for ease inhandling and manufacturing. Other sizes may be used, depending on thedesired therapeutic profile (e.g., the duration of sustained releasedesired, the effects, if any on biological activity, the ease inhandling, the degree or lack of antigenicity and other known effects ofthe PEG to a therapeutic polypeptide or analog).

The PEG molecules (or other chemical moieties) should be attached to thepolypeptide with consideration of effects on functional or antigenicdomains of the polypeptide. There are a number of attachment methodsavailable to those skilled in the art, e.g., EP 0 401 384, (coupling PEGto Granulocyte Colony Stimulating Factor (G-CSF)), Malik et al., (1992),Exp. Hematol. 20:1028-1035. This article reports on pegylation ofGranulocyte/Macrophage Colony Stimulating Factor (GM-CSF) using tresylchloride, which its disclosures is hereby incorporated by reference inits entireties. For example, PEG may be covalently bound through aminoacid residues via a reactive group, such as, a free amino or carboxylgroup. Reactive groups are those to which an activated PEG molecule maybe bound. The amino acid residues having a free amino group may includelysine residues and the N-terminal amino acid residues; those having afree carboxyl group may include aspartic acid residues glutamic acidresidues and the C-terminal amino acid residue. Sulfhydryl groups mayalso be used as a reactive group for attaching the PEG molecules.Preferred for therapeutic purposes is attachment at an amino group, suchas attachment at the N-terminus or lysine group.

The polypeptides herein can be chemically modified at the N-terminus.Using PEG as an illustration of the present composition, one may selectfrom a variety of PEG molecules (by molecular weight, branching, etc.),the proportion of PEG molecules to polypeptide (polypeptide) moleculesin the reaction mix, the type of pegylation reaction to be performed,and the method of obtaining the selected N-terminally pegylatedpolypeptide. The method of obtaining the N-terminally pegylatedpreparation (i.e., separating this moiety from other monopegylatedmoieties if necessary) may be by purification of the N-terminallypegylated material from a population of pegylated polypeptide molecules.Selective polypeptides chemically modified at the N-terminusmodification may be accomplished by reductive alkylation, which exploitsdifferential reactivity of different types of primary amino groups(lysine versus the N-terminal) available for derivitization in aparticular polypeptide. Under the appropriate reaction conditions,substantially selective derivitization of the polypeptide at theN-terminus with a carbonyl group containing polymer is achieved.

In any of the embodiments herein, a polypeptide of the present inventionmay be modified in its N-terminus Examples of N-terminus modificationsinclude an N-terminus methionine, N-terminus signal peptide, or aprosequence. An N-terminus methionine may be used for expression of apolypeptide recombinantly. A “signal sequence” or “presequence” refersto any sequence of amino acids bound to the N-terminal portion of apolypeptide herein (e.g., an amino acid sequence of SEQ ID NOs: 1-244,248-249, or any homolog or analog thereof), which may participate in thesecretion of the polypeptide. The term “prosequence” as used hereinrefers to a sequence of amino acids bound to the mature form of apolypeptide herein (e.g., an amino acid sequence of SEQ ID NOs: 1-244,248-249, or any homolog or analog thereof), which when removed resultsin the appearance of the “mature” form of the polypeptide (e.g., anamino acid sequence of SEQ ID NOs: 1-244, 248-249, or any homolog oranalog thereof). Preferably, a prosequence is autocleaved/cleaved bynaturally occurring enzymes, which are found at an area in which themature polypeptide needs to be active.

In some embodiments a polypeptide herein includes one or moreconservative substitutions. Such substitutions are selected from theTable I. Other known conserved substitutions may also be known to aperson of ordinary skill in the art.

In some embodiments, a polypeptide of the present invention is modifiedto be more resistant to proteolysis. For example, a polypeptidecomprising, consisting essentially of, or consisting of an amino acidsequence of (or encoded by) SEQ ID NOs: 1-244, 248-249, (any of theabove with a methionine at the N-terminus) or any homolog, analog orfragment thereof may include one or more peptide bonds in which the—CONH— peptide bond is modified and replaced by a non-cleavable bond,e.g., a (CH₂NH) reduced bond, a (NHCO) retro inverso bond, a (CH₂—O)methylene-oxy bond, a (CH₂—S) thiomethylene bond, a (CH₂CH₂) carba bond,a (CO—CH₂) cetomethylene bond, a (CHOH—CH₂) hydroxyethylene bond), a(N—N) bound, a E-alkene bond or also a —CH—CH-double bond.

In some embodiments, a polypeptide sequence herein is constructed in itsreverse sequence to prevent degradation. Examples of reverse sequenceinclude those of SEQ ID NO: SEQ ID NOs: 153-244.

2. Nucleic Acids

The present invention also provides for a nucleic acid that encodes anyof the polypeptides herein. For example, in some embodiments, thepresent invention relates to a nucleic acid sequence that encodes apolypeptide comprising, consisting essentially of, or consisting of anamino acid sequence selected from the group of SEQ ID NOs: 1-256, orselected from the group of SEQ ID NOs: 1-244, 248-249, or selected fromthe group of SEQ ID NOs SEQ ID NOs: 1-14 or 50-244, 248-249, or selectedfrom the group of SEQ ID NOs: 1, 2, and 15 or more preferably SEQ ID NO:1 (and any of the above with a methionine at the N-terminus) or afragment, homolog, or analog thereof.

For example, the present invention provides for a polynucleotidesequence comprising, consisting essentially of, or consisting of thefollowing sequence: [SEQ ID NO: 251: ttt ctg ccc tca]; SEQ ID NO: 252:ttt ctg ccc tca gaa ttt gga gta gac gta gac aga] or other nucleic acidsequence that encodes a peptide of the invention including all nucleicacid sequences permitted under codon degeneracy which is the divergencein the genetic code which permits variation of nucleotide sequencewithout effecting the amino acid sequence of an encoded polypeptide.Accordingly, the instant invention relates to any nucleic acid sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein.

Preferably any of the nucleotide sequences herein are preferablyisolated and/or purified.

The present invention also includes recombinant constructs comprisingone or more of the nucleotide sequences described herein. Suchconstructs comprise a vector, such as a plasmid or viral vector, intowhich a nucleic acid sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. Appropriate cloningand expression vectors for use with prokaryotic and eukaryotic hosts arealso described in Sambrook et al. (2001, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press).

Examples of such expression vectors include chromosomal, nonchromosomaland synthetic DNA sequences, e.g., derivatives of or Simian virus 40(SV40); bacterial plasmids; phage DNA; baculovirus; yeast plasmids;vectors derived from combinations of plasmids and phage DNA, viral DNAsuch as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However,any other vector may be used as long as it is replicable and viable inthe host. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, a nucleic acid sequenceencoding one of the polypeptides herein is inserted into an appropriaterestriction endonuclease site(s) by procedures known in the art. Suchprocedures and related sub-cloning procedures are deemed to be withinthe scope of those skilled in the art.

The nucleic acid sequence in the expression vector is preferablyoperatively linked to an appropriate transcription control sequence(promoter) to direct mRNA synthesis. Examples of such promoters include:the retroviral long terminal (LTR) or SV40 promoter, the E. coli lac ortrp promoter, the phage lambda PL promoter, and other promoters known tocontrol expression of genes in prokaryotic or eukaryotic cells or theirviruses. The expression vector also contains a ribosome binding site fortranslation initiation, and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Inaddition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell cultures, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate nucleic acid sequences asdescribed above, as well as an appropriate promoter or control sequence,may be employed to transform an appropriate host to permit the host toexpress the peptides herein (e.g., SEQ ID NOs: 1-256 more preferably SEQID NOs: 1-24, 50-244, and 248-249, or more preferably SEQ ID NOs: 1-2,or more preferably SEQ ID NO: 1). Such vectors can be used in genetherapy.

Examples of appropriate expression hosts include: bacterial cells, suchas E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such asyeast; insect cells such as Drosophila and Spodoptera frugiperda (Sf9);animal cells such as CHO, COS, HEK 293 or Bowes melanoma; adenoviruses;plant cells, etc. The selection of an appropriate host is deemed to bewithin the scope of those skilled in the art from the teachings herein.The invention is not limited by the host cells employed.

Thus, in some embodiment, the present invention relates to methods forproducing an analgesic peptide by transfecting a host cell with anexpression vector comprising a nucleotide sequence that encodes apeptide comprising, consisting essentially of, or consisting of an aminoacid sequence of (or encoded by) SEQ ID NOs: 1-244, 248-249, or any ofthe above with a methionine at the N-terminal, or any analgesic fragmentthereof, or any homolog or analog thereof. Such host cells are thencultured under a suitable condition, which allows for the expression ofsuch peptides.

The present invention further contemplates gene therapy using nucleicacids encoding one or more of the polypeptides herein (e.g., SEQ ID NOs:1-256 more preferably SEQ ID NOs: 1-24, 50-244, and 248-249, or morepreferably SEQ ID NOs: 1-2, or more preferably SEQ ID NO: 1) or ananalog or homolog thereof. Preferably, such gene therapy is targeted.Targeted gene therapy involves the use of vectors (organ- andtumor-homing peptides) that are targeted to specific organs or tissuesafter systemic administration. The vector consisted of a covalentconjugate of avidin and a monoclonal antibody to a receptor. Forexample, for delivery to the brain, a chimeric peptide had beenmonobiotinylated, to a drug transport vector. The vector consisted of acovalent conjugate of avidin and the OX26 monoclonal antibody to thetransferrin receptor. Owing to the high concentration of transferrinreceptors on brain capillary OX26 targets brain and undergoesreceptor-mediated transcytosis through the blood-brain barrier (Bickelet al., 1993, Proc. Nat. Acad. Sci. 90:2618-2622). Another example isvector-mediated delivery of opioid peptides to the brain (NIDA ResMonogr. 1995, 154:28-46).

In some embodiments, such nucleic acids are used to create a transgenicplant or animal, wherein the transgenic plant or animal is transgenicfor a polynucleotide of the present invention and expresses apolypeptide of the present invention. For introducing a nucleic acidencoding one or more of the peptides herein into a plant cell,introduction can be carried out by conventional gene engineeringtechniques, for example, Agrobacterium infection, electroporation intoprotoplasts, particle gun methods, and the like. In some embodiments,the nucleic acids above are introduced along with a second nucleic acidsequence or gene. In some embodiments, the second nucleic acid sequencecan act as a promoter, etc.

Preferably, the nucleic acid that is introduced into a plant cell isintegrated into a vector having a selection marker gene. For example,the nucleic acids encoding SEQ ID NOs: 1-256 more preferably SEQ ID NOs:1-24, 50-244, and 248-249, or more preferably SEQ ID NOs: 1-2, or morepreferably SEQ ID NO: 1, or a homolog or analog thereof can beintegrated into one of such vectors (e.g., pGEM-T and pBIN binaryvectors). The vectors are then introduced into a chromosome of a plantcell by homologous recombination (Fraley et al., 1983, Proc. Natl. Acad.Sci. USA, 80; 4803). Plant cells expressing the nucleic acids can thenbe selected. Alternatively, the nucleic acids can be introduced into aplant cell in a vector that it is operably linked to a promoter andoptionally a terminator both of which can function in the plant cell.

Non-limiting examples of promoters that can function in a plant cellinclude constitutive promoters derived from T-DNA such as nopalinesynthase gene promoter, octopine synthase gene promoter, etc., promotersderived from plant viruses such as 19S and 35S promoters derived fromcauliflower mosaic virus, etc., inductive promoters such asphenylalanine ammonia-lyase gene promoter, chalcone synthase genepromoter, pathogenesis-related polypeptide gene promoter, etc., and thelike.

Non-limiting examples of terminators that can function in a plant cellinclude terminators derived from T-DNA such as nopaline synthaseterminator, terminators derived from plant viruses such as terminatorsderived from garlic viruses GV1, GV2, and the like.

Plant cells into which such nucleic acids are introduced include planttissues, whole plants, cultured cells, seeds and the like. Examples ofthe plant species into which the genes are introduced includedicotyledones such as tobacco, cotton, rapeseed, sugar beet, Arabidopsisthaliana, canola, flax, sunflower, potato, alfalfa, lettuce, banana,soybean, pea, legume, pine, poplar, apple, grape, citrus fruits, nuts,etc.; and monocotyledones such as corn, rice, wheat, barley, rye, oat,sorghum, sugar cane, lawn, etc. The second gene may also be introducedinto such plant cells.

The transformant plant cells expressing one or more of the polypeptidesherein or homologs thereof can be obtained by culturing cells into whichthe gene is transferred in a selection culture medium corresponding to aselection marker joined to the locus on the gene, for example, a culturemedium containing a cell growth inhibitor, or the like, and isolating aclone capable of growing in the culture medium. Further, the selectionculture medium should also correspond to a selection marker joined tothe locus of the second gene when the altered form of enzymatic activityis also present in the transformant plant cells. Alternatively, theabove transformant plant cells can be selected by culturing plant cellsinto which the gene is introduced in a culture medium containing theweed control compound to which the resistance is given, and isolatingclones capable of growing in the culture medium.

The plant expressing the desired peptide can be obtained from thetransformant cells thus obtained by regenerating the whole plantaccording to a conventional plant cell culture method, for example, thatdescribed in Plant Gene Manipulation Manual, Method for ProducingTransgenic Plants, 1996, UCHIMIYA, Kodansha Scientific). Thus, thetransformed plants such as plant tissues, whole plants, cultured cells,seeds and the like can be obtained.

For example, rice and Arabidopsis thaliana expressing a gene encoding adesired peptide having the characteristics of having (i) anti-pyrogenic,(ii) anti-inflammatory, (iii) anti-neoplastic activity, or (iv)expressing resistance against pathogen, or (v) expressing developmentalchanges such as an increase in the number of flowers (e.g., SEQ ID NOs:1-256 more preferably SEQ ID NOs: 1-24, 50-244, and 248-249, or morepreferably SEQ ID NOs: 1-2, or more preferably SEQ ID NO: 1, or ahomolog or analog thereof) can be obtained according to the methoddescribed in Experimental Protocol of Model Plants, Rice and Mouse-EarCress Edition, Chapter 4 (1996, Supervisors: Shimamoto and Okada,Shujun-sha). Further, according to the method, soybean expressing ageneencoding a desired peptide by introducing the gene into soybeanadventitious embryo with a particle gun. Likewise, according to themethod described by Fromm et al., 1990, Bio/Technol., 8:838, cornexpressing the gene can be obtained by introducing the gene intoadventitious embryo with a particle gun. Wheat expressing the gene byintroducing the gene into sterile-cultured wheat immature scutellum witha particle gun according to a conventional method described by Takumi,1995, J. Breeding Soc., 44: Extra, 1:57. Likewise, according to aconventional method described by Hagio et al., 1995, J. Breeding Soc.,44; Extra, 1:67, barley expressing the gene encoding the abovepolypeptide can be obtained by introducing the gene intosterile-cultured barley immature scutellum with a particle gun.

Another embodiment is directed to fragments of the correspondent nucleicacid sequences, or the complement thereof, which may find use as, forexample, hybridization probes or as antisense oligonucleotides. Suchnucleic acid fragments are usually at least about 10 nucleotides inlength, preferably at least about 20 nucleotides in length, preferablyat least about 30 nucleotides in length, more preferably at least about40 nucleotides in length, yet more preferably at least about 50nucleotides in length, yet more, wherein in this context the term“about” means the referenced nucleotide sequence length plus or minus10% of that referenced length.

In some embodiments, the present invention relates to methods forisolating a gene or gene fragment encoding a peptide of the invention(SEQ ID NOs: 1-256 more preferably SEQ ID NOs: 1-24, 50-244, and248-249, or more preferably SEQ ID NOs: 1-2, or more preferably SEQ IDNO: 1, or a homolog or analog thereof) and homologs or analogs thereoffrom various organisms. Such gene or gene fragment can have (i)anti-pyrogenic, (ii) anti-inflammatory, (iii) anti-neoplastic activity,or (iv) expressing resistance against pathogen, or (v) expressingdevelopmental changes such as an increase in the number of flowers. Suchgene or gene fragment can be identified by performing PCR using genomicDNA or cDNA of an organism having the desired gene as a template andprimers produced on the basis of nucleotide sequences corresponding tothose about the N- and C-termini of the polypeptide to amplify thedesired gene. Further, genes encoding a peptide can be obtaineddifferent organisms (e.g., a clone, a plant, an animal, etc.). Forexample, first, a cDNA library is constructed by obtaining mRNA from anorganism and synthesizing cDNA by using the mRNA as template withreverse transcriptase and integrating the cDNA into a phage vector suchas ZAP II, etc. or a plasmid vector such as pUC, etc. The cDNA librarymay be introduced into Escherichia coli followed by subjecting acomplementation test to select clones containing the desired genederived from the desired organism. Further, for amplifying a DNAfragment containing at least a part of the desired gene, PCR can becarried out by using the above-constructed cDNA library as a templateand primers designed and synthesized on the basis of nucleotidesequences of the peptide. Screening of the cDNA library can be carriedout by using the DNA fragment thus obtained as a probe to selectpositive clones. The desired gene, i.e., a gene encoding a peptidesubstantially having at least one characteristics of (i) to, (v), can beconfirmed by determination of the nucleotide sequence of the selectedclone.

3. Antibodies

In another embodiment, the invention provides for antibodies thatspecifically bind to any of the polypeptides herein. For example, thepresent invention contemplates an antibody that specifically binds to apeptide of having an amino acid sequence comprising, consistingessentially of, or consisting of an amino acid sequence of SEQ ID NOs:1-244, 248-249, or more preferably SEQ ID NOs: 1-14 and 50-244, or morepreferably SEQ ID NOs: 1-2 or more preferably SEQ ID NO: 1, or any ofthe above with a methionine at the N-terminus, or any fragment, homolog,or analog thereof. The term “antibodies” is meant to include polyclonalantibodies, monoclonal antibodies, fragments thereof such as F(ab′)₂,and Fab fragments, as well as any recombinantly producedbinding-partners.

Antibody can be prepared by conventional methods, e.g. by immunizationof a human or of an animal, such as, for example, mouse, rat, guineapig, rabbit, horse, sheep, goat, chicken (see also Messerschmid, 1996,BIOforum, 11:500-502), and subsequent isolation of the antiserum; or byestablishing hybridoma cells and subsequent purification of the secretedantibodies; or by cloning and expression of the nucleotide sequences, ormodified versions thereof, which encode the amino acid sequences whichare responsible for the binding of the natural antibody to the antigenand/or hapten. Antibodies of the invention are in particular thoseantibodies which bind to a polypeptide comprising, consistingessentially of, or consisting of an amino acid selected from the groupconsisting of: SEQ ID NOs: 1-256, more preferably selected from thegroup consisting of SEQ ID NOs: 1-24, 50-244, and 248-29, morepreferably selected from the group consisting of SEQ ID NOs: 1-24,50-163, 248-249, more preferably selected from the group consisting ofSEQ ID NOs: 1-2, or more preferably SEQ ID NO: 1. In some embodiments,such polypeptide has less than 100 amino acid residues, 90, 80, 70, 60,50, 40, 30, 20, 10, 9, 8, 7, 6, 5, or 4 amino acid residues. In someembodiments, such polypeptide has between 4-100, 4-50, 4-40, 4-30, or4-20 amino acid residues. Preferably, such antibody selectively binds tothe amino acid sequence Phe-Leu-Pro-Ser-Glu-Phe-Gly-Val-Asp-Val-Asp-Arg(SEQ ID NO: 2) or more preferably amino acid sequence Phe-Leu-Pro-Ser(SEQ ID NO: 1)

The antibodies herein preferably specifically bind with a K_(a) ofgreater than or equal to about 10⁴ M⁻¹, preferably about 10⁵ M⁻¹, morepreferably about 10⁶ M⁻¹ and still more preferably about 10⁷ M⁻¹.Affinities of binding-partners or antibodies can be readily determinedusing conventional techniques, for example those described by Scatchardet al., 1949 (Ann. N.Y. Acad. Sci. 51:660) or, by surface plasmonresonance described by Wolff et al., 1993 (Cancer Res. 53:2560;BIAcore/Biosensor, Piscataway, N.J.), which are incorporated herein byreference for all purposes.

Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice or rats, using procedures that are well known in the art.In general, an isolated polypeptide of the invention (e.g., apolypeptide comprising, consisting essentially of, or consisting of anamino acid sequence of (or encoded by) SEQ ID NOs: 1-244, 248-249, orany of the above with an N-terminal methionine) that is appropriatelyconjugated, is administered to the host animal typically throughparenteral injection. The immunogenicity of the polypeptide may beenhanced through the use of an adjuvant, for example, Freund's completeor incomplete adjuvant. Following booster immunizations, small samplesof serum are collected and tested for reactivity to the polypeptide.Examples of various assays useful for such determination include thosedescribed in Antibodies: A Laboratory Manual, Harlow and Lane (eds.),Cold Spring Harbor Laboratory Press, 1988; as well as procedures such ascountercurrent immuno-electrophoresis (CIEP), radioimmunoassay (RIA),radioimmunoprecipitation, enzyme-linked immunosorbent assays (ELISA),dot blot assays, and sandwich assays, see, e.g., U.S. Pat. Nos.4,376,110 and 4,486,530, or other similar assays known in the art.

Monoclonal antibodies specific for a desired polypeptide antigen (suchas the peptides described herein) may be readily prepared usingwell-known procedures, see for example, the procedures described inCurrent Protocols in Immunolog (Wiley & Sons, NY, Coligan et al., eds.,1994; U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439 and 4,411,993;Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, 1980, Plenum Press, Kennett et al., eds.). Briefly, the hostanimals, such as mice are injected intraperitoneally at least once, andpreferably at least twice at about three-week intervals with isolatedand purified polypeptide herein (e.g., a polypeptide comprising,consisting essentially of, or consisting of an amino acid sequence of(or encoded by) SEQ ID NOs: 1-244, 248-249, or, conjugated polypeptideherein, optionally in the presence of adjuvant. Mouse sera are thenassayed by conventional dot blot technique or antibody capture (ABC) todetermine which animal is most suitable as a source of splenocytes forfusion to a myeloma partner cell-line. Approximately 2-3 weeks later,the mice are given an intravenous boost of the polypeptide. Mice arelater sacrificed and spleen cells fused with commercially availablemyeloma cells, such as Ag8.653 (ATCC), following established protocols.Briefly, the myeloma cells are washed several times in media and fusedto mouse spleen cells at a ratio of about three spleen cells to onemyeloma cell. The fusing agent can be any suitable agent used in theart, for example, PEG. The cell suspension containing fused cells isplated out into plates containing media that allows for the selectivegrowth of the fused cells. The fused cells can then be allowed to growfor approximately eight days. Supernatants from resultant hybridomas arecollected and added to a plate that is first coated with goat anti-mouseIg. Following washes, a label, such as, ¹²⁵I-conjugated polypeptide(e.g., a polypeptide comprising, consisting essentially of, orconsisting an amino acid sequence of (or encoded by) SEQ ID NOs: 1-244,248-249, or any of the above with an N-terminal methionine) is added toeach well followed by incubation. Positive wells can be subsequentlydetected by autoradiography. Positive clones can be grown in bulkculture and supernatants are subsequently purified over a Polypeptide Acolumn (Pharmacia).

The monoclonal antibodies of the invention can be produced usingalternative techniques, such as those described by Alting-Mees et al.,1990 (Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas, Strategies, Mol. Biol. 3:1-2469). Similarly, bindingpartners can be constructed using recombinant DNA techniques toincorporate the variable regions of a gene that encodes a specificbinding antibody. Larrick et al. describe such technique in Biotechnol,7:394, 1989.

Other types of antibodies may be produced using the information providedherein in conjunction with the state of knowledge in the art. Forexample, antibodies that have been engineered to contain elements ofhuman antibodies that are capable of specifically binding any of thepeptide or to a polypeptide containing the peptide sequence herein arealso encompassed by the invention. An additional method for selectingantibodies that specifically bind to a polypeptide, peptide or fragmentthereof is by phage display, e.g., Winter et al., 1994, Annu. Rev.Immunol 12: 433; Burton et al., 1994, Adv. Immunol 57:191. Human ormurine immunoglobulin variable region gene combinatorial libraries maybe created in phage vectors that can be screened to select Ig fragments(Fab, Fv, sFv, or multimers thereof) that bind specifically to apolypeptide, peptide, or fragment thereof. See, e.g., U.S. Pat. No.5,223,409; Huse et al., 1989, Science:1275; Kang et al., 1991 Proc.Natl. Acad. Sci. USA 88:4363; Hoogenboom et al., 1992, J. Molec. Biol.227:381; Schlebusch et al., 1997, Hybridoma 16:47, and references citedtherein. For example, a library containing a plurality of polynucleotidesequences encoding Ig variable region fragments may be inserted into thegenome of a filamentous bacteriophage, such as M13 or a variant thereof,in frame with the sequence encoding a phage coat polypeptide, forinstance, gene III or gene VIII of M13, to create an M13 fusionpolypeptide. A fusion polypeptide may be a fusion of the coatpolypeptide with the light chain variable region domain and/or with theheavy chain variable region domain. Once isolated and purified, theantibodies may be used to detect the presence of a polypeptide, or apeptide of the present invention in a sample using established assayprotocols. Further, the antibodies of the invention may be usedtherapeutically to bind to the peptides of the invention and alter theiractivity in vivo.

Formulations

Any of the composition herein may be formulated into pharmaceutical,veterinary, cosmetic and/or agricultural formulations for administrationto an organism.

Typically such formulations will include one or more acceptablecarriers, excipients, or diluents. Pharmaceutically acceptable carriersfor therapeutic use are well known in the pharmaceutical art, and aredescribed, e.g., in Remington's Pharmaceutical Sciences, Gennaro, A R,ed., 20th edition, 2000: Williams and Wilkins PA, USA. which isincorporate herein by reference for all purposes. Agriculturallyacceptable carriers for therapeutic or prophylactic treatment of plantsare also known in the art. Cosmetic and veterinary excipients are alsoknown in the art.

For example, the compositions herein may be combined with one or morenatural or synthetic, organic or inorganic material to facilitate theirapplication into the plant. Such a carrier will generally be inert andacceptable in agriculture. Such carrier can be solid (e.g., clays,natural or synthetic silicates, silica, resins, waxes, or solidfertilizers) or liquid (e.g., water, alcohols, ketones, petroleumfractions, aromatic or paraffinic hydrocarbons, chlorinatedhydrocarbons, or liquefied gases).

A pharmaceutical or agricultural formulation can also contain any kindof other compatible ingredients such as, for example, protectivecolloids, adhesives, thickening agents, thixotropic agents, penetratingagents, stabilizing agents, sequestering agents, fertilizers,anti-freeze agents, repellents, color additives, corrosion inhibitors,water-repelling agents, UV-stabilizers, pigments, dyes or polymers.

In some embodiments, the compositions herein may be formulated as a saltand be formed with many acids, including but not limited to,hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, ata pH range of 4.5 to 5.5 that is combined with buffer prior to use.After pharmaceutically and physiologically acceptable compositions havebeen prepared, they can be placed in an appropriate container andlabeled for treatment of an indicated condition.

While any suitable carrier known may be employed in a pharmaceuticalformulation of this invention, the type of carrier will vary dependingon the mode of administration and whether a sustained release isdesired. Routes of delivery may include oral, inhaled, buccal,intranasal, and transdermal routes, as well as novel delivery systemssuch as the protective liposomes for oral delivery of peptides.

For agricultural uses, formulations are preferably in a liquid or sprayor any other dry formulations.

For parenteral administration, such as subcutaneous injection, thecarrier preferably comprises water, saline, alcohol, a fat, a wax or abuffer.

For oral administration, a carrier preferably comprises of carbohydrateor polypeptide fillers, such as sugars, including lactose, sucrose,mannitol, or sorbitol; starch from corn, wheat, rice, potato, or otherplants; cellulose, such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gumsincluding arabic and tragacanth; and polypeptides such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, alginicacid, or a salt thereof, such as sodium alginate. If desirable, the drugcan be delivered in nanocapsules that would protect against proteolysisby proteases. Such carriers enable the compositions herein to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions, and the like, for ingestion by the patient.Pharmaceutical preparations for oral use can be obtained through acombination of active compounds with solid excipient, suiting mixture isoptionally grinding, and processing the mixture of granules, afteradding suitable auxiliaries, if desired, to obtain tablets or drageecores. As the composition may be peptide, such peptides are preferablyput into a liposomal formulation to avoid degradation.

Preferably the pharmaceutical formulations herein are administration byintravenous injection or by local applications (e.g., topical orsubdermal).

Formulations for topical administration can use a carrier that is asolution, emulsion, and ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, PEGs,beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device.

Biodegradable microspheres (e.g., polylactic galactide) may also beemployed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109. In this regard, itis preferable that the microsphere be larger than approximately 25 μm.

Pharmaceutical compositions may also contain diluents such as buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, polypeptides, amino acids,carbohydrates including glucose, sucrose or dextrins, chelating agentssuch as EDTA, glutathione and other stabilizers and excipients. Neutralbuffered saline or saline mixed with nonspecific serum albumin areexemplary appropriate diluents. Preferably, product is formulated as alyophilizate using appropriate excipient solutions (e.g., sucrose) asdiluents.

Pharmaceutically acceptable formulations include compositions whereinthe active ingredients (e.g., a polypeptide comprising of, consistingessentially of, or consisting of an amino acid sequence of (or encodedby) SEQ ID NOs: 1-244, 248-249, or any analog, or homolog thereof) arecontained in an effective dose to achieve the intended purpose. Thedetermination of an effective amount or dosage is well within thecapability of those skilled in the art. Typically, an effective dose ofa polypeptide of the present invention (e.g., a polypeptide comprisingof, consisting essentially of, or consisting of an amino acid sequenceof (or encoded by) SEQ ID NOs: 1-244, 248-249, or any analog or homologthereof) for systemic administration is between about 0.001 μg to about100 g, or more preferably between about 0.01 μg to about 50 g, or morepreferably between about 0.1 μg to about 1 g, or more preferably betweenabout 1 mg to about 500 mg per dose. For topical administration, thecompositions herein may be delivered at dosage up to about 99, 95, 90,80, 70, 60, 50, 40, or 30% w/w of the composition.

In some embodiments, the therapeutic effective dosages of SEQ ID NOs:1-256 are the serum concentrations that in the range of 1-1000 mg/L,5-500 mg/L, or 10-100 mg/L, or 10-20 mg/L or the active ingredient.

Any of the compositions herein may be co-formulated or co-administeredwith a second therapeutic agent. Examples of therapeutic agents include,but are not limited to, analgesic, antipyretic medicaments (feverreducers), anesthetics, anti-rheumatic agents, anti-inflammatory agents,antidepressants, anti-neoplastic agents, antimicrobial agents (e.g.,antibiotics, antiviral agents, and antifungal agents), pesticides,herbicides, angiogenic agents, anti-angiogenic agents, inhibitors ofneurotransmitters or neurotransmitters, any agent known to treatneurodegenerative conditions and wound healing, and combinationsthereof.

The concentration of an active ingredient in the composition of thepresent invention, as applied to plants is preferably within the rangeof 0.01 to 30.0% by weight, especially 0.1 to 30% % by weight. In aprimary composition, the amount of active ingredient can vary widely andcan be, for example, from 5 to 95% by weight of the composition.

For any of the compositions herein, the therapeutically effective dosecan be estimated initially either in cell culture assays, e.g., ofneoplastic cells, or in animal models, usually mice, rabbits, dogs, orpigs. The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans. Those of ordinary skill in the art are well able to extrapolatefrom one model (be it an in vitro or an in vivo model). Atherapeutically effective dose refers to that amount of activeingredient, for example a polypeptide comprising of, consistingessentially of, or consisting of an amino acid sequence of (or encodedby) SEQ ID NOs: 1-244, and 248-249, or any fragment, analog, or homologthereof, which ameliorates the symptoms or condition. Therapeuticefficacy and toxicity may be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED₅₀ (thedose therapeutically effective in 50% of the population) and LD₅₀ (thedose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the ratio, LD₅₀/ED₅₀. Pharmaceutically and physiologicallyacceptable compositions, which exhibit large therapeutic indices, arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration. The practitioner, in light of factors related to thesubject that requires treatment, will determine the exact dosage. Dosageand administration are adjusted to provide sufficient levels of theactive moiety or to maintain the desired effect. Factors, which may betaken into account, include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutically and physiologically acceptable compositions maybeadministered every 3 to 4 days, every week, or once every two weeksdepending on half-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.001 μg to 100 g, up to a totaldose of about 1 g, depending upon the route of administration. Guidanceas to particular dosages and methods of delivery is provided in theliterature and generally available to practitioners in the art. Thoseskilled in the art will employ different formulations for nucleotidesthan for polypeptides or their inhibitors. Similarly, delivery ofpolynucleotides or polypeptides will be specific to particular cells,cell types, organism being treated, conditions, locations, etc.

For example, for the prevention or treatment of pain, the appropriatedosage of an anti-pain medicament will depend on the type of conditionto be treated, as defined above, the severity and course of the disease,whether the agent is administered for preventive or therapeutic purposesor, as a combination with other drugs, previous therapy, the patient'sclinical history and response to the agent, and the discretion of theattending physician. The agent is suitably administered to the patientat one time or over a series of treatments.

Animal experiments provide reliable guidance for the determination ofeffective doses for human therapy. Interspecies scaling of effectivedoses can be performed following the principles laid down by Mordenti,J. and Chappell, W. “The use of interspecies scaling in toxicokinetics”in Toxicokinetics and New Drug Development, Yacobi et al., eds.,Pergamon Press, New York 1989, pp. 42-96. For example, depending on thetype and severity of the disease, about 0.001 mg/kg to 1000 mg/kg of atherapeutic agent is an initial candidate dosage for administration tothe patient, whether, for example, by one or more separateadministrations, or by continuous infusion. A typical daily dosage mightrange from about 1 g/kg to 100 g/kg or more, depending on the factorsmentioned above. For local administration or topical administrationlower dosage may be required. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays. Guidance as toparticular dosages and methods of delivery is provided in theliterature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; orU.S. Pat. No. 5,225,212. It is anticipated that different formulationswill be effective for different treatment compounds and differentdisorders, that administration targeting one organ or tissue, forexample, may necessitate delivery in a manner different from that toanother organ or tissue.

The compositions may be administered in the form of a solid, liquid, gelor gas (aerosol). The pharmaceutical composition is formulated so as toallow the active ingredients contained therein to be bioavailable uponadministration of the composition to a patient. Compositions that willbe administered to a patient take the form of one or more dosage units,where for example, a tablet may be a single dosage unit, and a containerof one or more compounds of the invention in aerosol form may hold aplurality of dosage units.

For oral administration, an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed.

The composition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred compositions contain, inaddition to the compositions herein one or more of a sweetening agent,preservatives, dye/colorant and flavor enhancer. In a compositionintended to be administered by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent may be included.

Injectable formulations of the compositions herein are preferablysterile. Means for achieving sterility are well known in the art.

For delivery to the dermis and/or epithelium, dermal patches anddelivery systems, utilizing active or passive transdermal deliverycarriers may be prepared suing well known methods and materials,including, for example, microporous membranes, silicon polymers anddiffusion matrixes. Such materials and methods are described, forexample, in: Remington's Pharmaceutical Sciences, supra.

For use in plants, the compositions of the invention are generallyapplied to seeds, plants or their habitat. Thus, the compositions hereincan be applied directly to the soil before, at or after drilling so thatthe presence of active compound in the soil can control the growth ofpathogens, which may attack the seeds. When the soil is treated directlywith a composition herein, it can be applied in any manner which allowsit to be intimately mixed with the soil, e.g., by spraying, bybroadcasting a solid form of granules, or by applying the activeingredient at the same time as drilling by inserting it in the samedrill as the seeds. A suitable application rate is within the range offrom 0.005 to 1000 g per hectare, more preferably from 0.10 to 500 g perhectare.

Alternatively, the active compounds can be applied directly to a plantby, for example, spraying or dusting either at the time when a pathogenhas begun to appear on the plant or before the appearance of a pathogenas a protective measure. In both such cases the preferred mode ofapplication is by foliar spraying. It is generally important to obtaingood control of pathogens in the early stages of plant growth, as thisis the time when the plant can be most severely damaged.

The spray or dust can further contain a pre- or post-emergence herbicideif this is thought necessary. Sometimes, it is practicable to treat theroots, bulbs, tubers or other vegetative propagule of a plant before orduring planting, for example, by dipping the roots in a suitable liquidor solid composition. When the active compound is applied directly tothe plant a suitable rate of application is from 0.002 to 5 kg perhectare, preferably from 0.005 to 1 kg per hectare, or more preferablyfrom 0.01 to 0.05 kg per hectare.

Conditions Affecting Animals and Plants

In some aspects, the present invention relates to uses of compositionssuch as the peptides disclosed herein (Table V) for modulating,preventing, or treating condition(s) in an organism. Such organisms canbe animals and/or plants. Animals are preferably domesticated animals orhumans. Plants are preferably crops such as wheat, barley, rice, corn,sugar, or soy; vegetables or fruits, such as apples, pears, citrusfruits, berries and nuts; and/or flowering plants such as roses,gardenias, orchids, carnations, bird of paradise, etc. But other plantsor parts of plants are also contemplated herein (e.g., trees for lumber,such as fir, redwoods, pine, etc.)

The conditions that are modulated, prevented, or treated by thecompositions herein can be broadly classified as metabolic ormitochondrial conditions. More specifically, such conditions are e.g.,thermogenic or pyrogenic conditions. Such conditions can be associatedwith, for example, pain, temperature regulation, inflammation,neoplastic growth (e.g., cancer), innate immune response activation andability to fight parasites and pathogens, skin and dermatologicalconditions, diabetes related disorders, wound healing, undesirable drugside effects, and neurological and neurodegenerative conditions.

Such conditions can occur in a cell, group of cells, or an entireorganism to be treated herein.

1. Pain.

In one aspect the present invention relates to treatment of pain.Examples of pain conditions contemplated by the invention include, butare not limited to, headaches (e.g., trigeminal neuralgia, sinusitis,cluster headaches, migraines, etc.), low back pain, cancer pain,arthritis pain, muscle spasm pain (muscle cramps), bone pain, painresulting from burns, pain associated with bumps, pain associated withbruises, inflammatory pain (from an infection or arthritic disorder),pain from obstructions, myofascial pain, pain from nerve trauma(dystrophy/causalgia), phantom limb pain, entrapment neuropathy (e.g.,carpal tunnel syndrome), peripheral neuropathy, and pain from wounds,e.g., surgical, accidential, or self-inflicted wounds.

The pathophysiology of pain can be broadly divided into threecategories: (i) nociceptive pain, (ii) neuropathic pain, and (iii)idiopathic pain. (Willis, W. D., 1985, The Pain System. The Neural Basisof Nociceptive Transmission in the Mammalian Nervous System. Pain andHeadache, vol. 8, Gildenberg PL (Ed.) Karger Publishers, New York).

Nociceptive pain is the result of receptor stimulation by tissue injury.It involves the normal activation of the nociceptive system by noxiousstimuli. Examples of nociceptive pain include sprains, bone fractures,burns, bumps, bruises, inflammation (from an infection or arthriticdisorder), obstructions, myofascial pain (which may indicate abnormalmuscle stresses) headaches, low back pain, cancer pain, and arthritispain. In some embodiments, the compositions herein are used to preventor treat nociceptive pain. Preferably, such compositions are an aminoacid sequence of (or encoded by) SEQ ID NOs: 1-244, 248-249. In someembodiments, second therapeutic agents such as NSAIDs (Non-SteroidalAnti-Inflammatory Drugs), and/or opioids can be used in combination withthe compositions herein to treat nociceptive pain.

Thus, in one aspect, the present invention relates to uses of thecompounds herein for treating nociceptive pain. Such methods involveadministering one or more of the compositions herein to a subjectsuffering or susceptible of suffering nocicpetive pain. Such compositionpreferably a polypeptide comprising, consisting essentially of, orconsisting of an amino acid sequence selected from the group of SEQ IDNOs: 1-244, 248-249, or from the group of SEQ ID NOs: 1-14, 50-152, and248-249 or SEQ ID NOs: 1-2 or SEQ ID NO: 1. In one embodiment, suchcomposition comprises a nucleic acid sequence that encodes a polypeptidecomprising, consisting essentially of, or consisting of an amino acidsequence selected from the group of SEQ ID NOs: 1-244, 248-249, or fromthe group of SEQ ID NOs: 1-14, 50-152, and 248-249 or SEQ ID NOs: 1-2 orSEQ ID NO: 1. In one embodiment, such composition comprises an antibodythat specifically binds a polypeptide comprising, consisting essentiallyof, or consisting of an amino acid sequence selected from the group ofSEQ ID NOs: 1-244, 248-249, or from the group of SEQ ID NOs: 1-1450-152, and 248-249 or SEQ ID NOs: 1-2 or SEQ ID NO: 1.

The second category of pain, neuropathic pain, is the result of aninjury or malfunction in the peripheral or central nervous system.Examples of neuropathic pain include post herpetic (or post-shingles)neuralgia, reflex sympathetic dystrophy/causalgia (nerve trauma),components of cancer pain, phantom limb pain, entrapment neuropathy(e.g., carpal tunnel syndrome), and peripheral neuropathy most commonlycaused by diabetes or chronic alcohol use.

Neuropathic pain is often triggered by an injury, but this injury may ormay not involve actual damage to the nervous system. For example, nervescan be infiltrated or compressed by tumors, strangulated by scar tissue,or inflamed by infection, which may cause neuropathic pain. Neuropathicpain may persist for months or years beyond the apparent healing of anydamaged tissues. Therefore, neuropathic pain is frequently chronic, notfully reversible, and tends to have a less robust response to treatmentwith opioids, but may respond to drugs such as anticonvulsants(carbamazepine and valproic acid, and gabapentin) and neuromodulatingdrugs (including tricyclic antidepressants, such as amitriptyline,imipramine, and desipramine).

The present invention contemplates uses of the compositions herein fortreatment of neuropathic pain. In particular, the present inventionincludes methods for treating neuropathic pain in a subject byadministering one or more of the compositions herein to the subject in atherapeutically effective amount to treat or prevent neuropathic pain.In preferred embodiment, the composition herein used to treatneuropathic pain preferably comprise or consist essentially orconsisting of a polypeptide comprising, consisting essentially of, orconsisting of an amino acid sequence selected from the group of SEQ IDNO: 1-2, or from the group of SEQ ID NOs: 1-14 and 50-244, or SEQ IDNOs: 1-2 or SEQ ID NO: 1. In one embodiment, such composition comprisesa nucleic acid sequence that encodes a polypeptide comprising,consisting essentially of, or consisting of an amino acid sequenceselected from the group of SEQ ID NOs: 1-244, 248-249, or from the groupof SEQ ID NOs: 1-14 and 50-244, or SEQ ID NOs: 1-2 or SEQ ID NO: 1. Inone embodiment, such composition comprises an antibody that specificallybinds a polypeptide comprising, consisting essentially of, or consistingof an amino acid sequence selected from the group of SEQ ID NO: 1-244,248-249, or from the group of SEQ ID NOs: 1-14 and 50-244, or SEQ IDNOs: 1-2 or SEQ ID NO: 1.

The third category of pain, idiopathic pain, is a diagnosis of exclusionin which a patient suffers pain for longer than 6 months for which thereis no physical cause and no specific mental disorder. Examples ofidiopathic pain include, but are not limited to, arthritis,fibromyalgia, chronic fatigue syndrome, irritable bowel syndrome,interstitial cystitis, vulvadynia, carpal tunnel syndrome, etc.

In one aspect, the present invention relates to uses of the compoundsherein for treating idiopathic pain. Such methods involve administeringone or more of the compositions herein to a subject suffering orsusceptible of suffering idiopathic pain. Such composition preferablyinclude a polypeptide comprising, consisting essentially of, orconsisting of an amino acid sequence selected from the group of SEQ IDNO: 1-244, 248-249, or from the group of SEQ ID NOs: 1-14 and 50-244, orSEQ ID NOs: 1-2 or SEQ ID NO: 1. In one embodiment, such compositioncomprises a nucleic acid sequence that encodes a polypeptide comprising,consisting essentially of, or consisting of an amino acid sequenceselected from the group of SEQ ID NOs: 1-244, 248-249, or from the groupof SEQ ID NOs: 1-14 and 50-244, or SEQ ID NOs: 1-2, or SEQ ID NO: 1. Inone embodiment, such composition comprises an antibody that specificallybinds a polypeptide comprising, consisting essentially of, or consistingof an amino acid sequence selected from the group of SEQ ID NOs: 1-244,248-249, or from the group of SEQ ID NOs: 1-14 and 50-244, or SEQ IDNOs: 1-2 or SEQ ID NO: 1.

Any of the compositions herein can be administered either singly or incombination with a second therapeutic agent such as an analgesic painreliever or anti-inflammatory. In some embodiments, the secondtherapeutic agent is co-formulated with one or more of the compositionsherein.

According to the National Drug Classification (NDC), analgesics can becategorized in to the following group: general analgesic, narcoticanalgesic, non-narcotic analgesic, anti-arthritics,anti-migraine/headache, central pain syndrome, NSAID, anti-pyretic, andanti-menstrual pain products. These categories can be combined intobroader categories of analgesics entitled: narcotic analgesics,non-narcotic analgesics, and NSAIDs.

The present invention relates to a pharmaceutical formulation comprisingthe combination of the polypeptides herein with one or more analgesicagents selected from the group consisting of general analgesic, narcoticanalgesic, non-narcotic analgesic, anti-arthritics,anti-migraine/headache, central pain syndrome, NSAID, anti-pyretic, andanti-menstrual pain products. The present invention also relates to apharmaceutical formulation comprising the combination of thepolypeptides herein with one or more analgesics selected from the groupconsisting of narcotic analgesics, non-narcotic analgesics, and NSAIDs.The present invention also relates to methods of treating a subjectsuffering from pain (e.g., nociceptive pain, neuropathic pain, andidiopathic pain) comprising administered to said subject the one or morecompositions herein and the one or more analgesics described herein(either separately or in combination, as a co-formulation or in twoseparate formulations). Preferably the polypeptides comprise, consistessentially of, or consist of an amino acid sequence selected from thegroup of SEQ ID NOs: 1-14 and 50-244, or SEQ ID NOs: 1-2 or SEQ ID NO:1.

Examples of narcotic analgesics include, but are not limited to,Alfentanil; Allylprodine; Alphaprodine; Amiphenazole, Anileridine,Benzoylhydrazone, Benzylmorphine, Benzitramide, Nor-Binaltorphimine,Bremazocine; Bupremorphine; Butorphanol (Stadol); Clonitazene; Codeine;CTOP; Cyclazocine; DAMGO; Desomorphine; Dextromoramide; Dezocine;Diampromide; Dihydrocodeine; Dihydrocodeine enol acetate;Dihydromorphine; Dimenoxadol; Dimepheptanol; Dimethylthiambutene;Dioxaphetyl Butyrate; Dipipanone; Diprenorphine; DPDPE; Eptazocine;Ethoheptazine; Ethylketocyclazocine; Ethylmethylthiambutene;Etonitazene; Etorphine; Fentanyl (Sublimaze, Duragesic); Hydrocodone;Hydromorphone (Dilaudid); Hydroxypethidine; Isomethadone; ketobemidone;Levorphanol; Levallorphan; Lofentanil; Loperamide; Meperidine (Demerol);Meptazinol; Metazocaine; Methadone (Dolophine); Metopon; Morphine(Roxanol); Myrophine; Nalbuphine; Nalmefene; Nalorphine; Naloxone;Naltrindole; Naltrexone; Narceine; Nicomorphine, Norlevorphanol;Normethadone; Normorphine; Norpipanone; Opium; Oxycodone (OxyContin);Oxymorphone; Papavereturn; Papaverine; Pentazocine; Phenadoxone;Phenazocine; Phenoperidine; Piminodine; Pirtramide; Proheptazine;Promedol; Propiram; Propoxyphene (Darvon); Remifentanil; Spiradoline;Sufentanil; Tilidine; U50,488; and U69,593. Some products arecombination drugs; codeine/acetaminophen (APAP; Tylenol #3);hydrocodone/acetaminophen (Vicodin); Oxycodone/ASA (Percodan);oxycodone/APAP (Percocet); propoxyphene/ASA (Darvon Compound);propoxyphene/napsylate (Darvocet-N); hydrocodone/ibuprofen (Vicoprofen);pentazocine/naloxone (Talwin-Nx).

Examples of non-narcotic analgesics, include, but are not limited to,Acetaminophen (Paracetamol; Tylenol); aspirin (acetylsalicylic acid;Anacin, Ascriptin, Bayer, Bufferin, Ecotrin, Excedrin); AminobenzoicAcid; Capsaicin (Zostrix and Zostrix-HP); Carbaspirin Calcium; Cholineand Magnesium Salicylates (CMT, Tricosal, Trilisate); Choline Salicylate(Arthropan); Etanercept; Fluprednisolone; Gold sodium Thiomalate; GoldSodium Thiosulfate; Hyaluronic Acid; Homomethyl Salicylate; Leflunomide;Magnesium Salicylate (Arthritab, Bayer Select, Doan's Pills, Magan,Mobidin, Mobogesic); Menthol; Methorexate; Octyl Salicylate;Oxyphenbutazone; Phenyl Salicylate; Phenylbutazone; Prednisolone;Salicylamide; Salsalate (Amigesic, Anaflex 750, Disalcid, Marthritic;Mono-Gesic, Salflex, Salsitab); Sodium Hyaluronate; Sodium Salicylate;o-Acetylsalicyloyl Chloride; Sodium Thiosalicylate (Thiocyl); Tramadol;Triamcinilone; Triethanolamine Salicylate (Trolamine); Zomepirac. Someproducts, such as Excedrin, are combination drugs (Excedrin isacetaminophen, ASA, and caffeine). Other non-narcotic gabapentin(Neurontin); lamotrigine and, anti-convulsants and tricyclicanti-depressants such as carbamazepine, pregabalin and duloxetine.

Examples of NSAIDS include, but are not limited to, Bromfenac Sodium;Celecoxib (Celebrex); Diclofenac Potassium (Cataflam); Diclofenac Sodium(Voltaren, Voltaren XR); Diclofenac Sodium with misoprostol (Arthrotec);Diflunisal (Dolobid); Etodolac (Lodine, Lodine XL); Etadolac; Fenoprofencalcium (Nalfon); Flurbiprofen (Ansaid); Ibuprofen (Motrin, Advil,Nuprin); Indomethacin (Indocin, Indocin SR); Ketoprofen (Actron, Orudis,Orudis KT, Oruvail); Meclofenamate Sodium (Meclomen); Mefenamic acid(Ponstel); Meloxicam (Mobic); Nabumetone (Relafen); Naproxen (Naprosyn,Naprelan, Alleve, Anaprox); Oxaprozin (Daypro); Piroxicam (Feldene);Piroxicam (Feldene); Rofecoxib (Vioxx); Sulindac (Clinoril); Suprofen;Tolmetin Sodium (Tolectin); Valdecoxib (Bextra).

Furthermore, there are various naturally occurring and synthetic opioidsthat can be used to treat pain. See Table II below.

TABLE II List of opioid peptides Enkephalins [Leu] - enkephalin YGGFL(SEQ ID NO: 15) [Met] - enkephalin YGGFM (SEQ ID NO: 16) RimorphinYGGFLRRQFKVVT (SEQ ID NO: 248) LeumorphinKYPKRSSEVAGEGDGDSMGHEDLYKRYGGFLRRIRPKLKWDNQKRYGGFLRRQFKVVTRSQEDPNAYSGELFDA (SEQ ID NO: 249) Endorphinsα-Neoendorphin YGGFLRKYPK (SEQ ID NO: 17) β-Neoendorphin YGGPLRKYP(SEQ ID NO: 18) β-human-Endorphin YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE(SEQ ID NO: 19) α-human-Endorphin YGGFMTSEKSQTPLVT (SEQ ID NO: 20)Dynorphins DynorphinA YGGFLRRIRPKLKWDNQ (SEQ ID NO: 21) Dynorphin BYGGFLRRQFKVVT (SEQ ID NO: 22) Endomorphins Endomorphin-1 YPTF(SEQ ID NO: 23) Endomorphin-2 YPFF (SEQ ID NO: 24) Synthetic peptides[D-Ala², N-Me-Phe⁴, Gly⁵-0l]- [Tyr-D-Ala-Gly-N-Methyl-Phe-Gly-olenkephalin (DAMGO; DAGO) (SEQ ID NO: 25) D-Pen^(2,5)]- enkephalin (DPDPE) [Tyr-D-Pen-Gly-Phe-D-Pen (SEQ ID NO: 26)D-Pen^(2,5)] - enkephalin (pCl-DPDPE) Tyr-D-Pen-Gly-D-Chloro-Phe-D-Pen(SEQ ID NO: 27) [Pen², Pen⁵]-enkephalin (DPLPE) Tyr-D-Pen-Gly-Pen-Pen(SEQ ID NO: 28) [D-Ser², D-Leu⁵] - enkephalin-ThrTyr-D-Ser-Gly-Phe-Leu-Thr (DSLET) (SEQ ID NO: 29) [D-Ala², D-Leu⁵]- enkephalin Tyr-D-Ala-Gly-Phe-D-Leu (DADLE) (SEQ ID NO: 30)Met-enkephalin-Arg-Phe (MERF) Tyr-Gly-Gly-Phe-Met-Arg-Phe(SEQ ID NO: 31) CTOP D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr (SEQ ID NO: 32)Ac-RYYRIK Ac-Arg-Tyr-Arg-Ile-Lys (SEQ ID NO: 33)([D-Arg2, Lys4+-Dermorphin1)- Tyr-D-Arg-Phe-Lys amide(DALDA)(SEQ ID NO: 34) (D-Ala², N-Methyl-Phe⁴, Met(O)5-ol] -Tyr-D-Ala-Gly-N-Methyl-Phe-Gly-ol enkephalin (FK-33824) (SEQ ID NO: 35)[D-Ala², Leu⁵, Cys⁶] - enkephalin Tyr-D-Ala-Gly-Phe-D-Leu-D-Cys (DALCE)(SEQ ID NO: 36) [D-Ala² Glu⁴] - Deltorphin IITyr-D-Ala-Phe-Glu-Val-Val-Pro Gly-amide (SEQ ID NO: 37)[D-Ala²]Deltorphin 1 Tyr-D-Ala-Phe-Asp-Val-Val-Gly (SEQ ID NO: 38)PL-017 Tyr-Pro-Methyl-Phe-D-Pro (SEQ ID NO: 39) ICI 174,8674N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (SEQ ID NO: 40) Others Morphiceptin YPFP(SEQ ID NO: 41) Nociceptin orphanin FQ FGGFTGARKSARKLANQ (SEQ ID NO: 42)Nocistatin TEPGLEEVGEIEGQKQLQ (SEQ ID NO: 43)Neuropeptide AF human (NPAF) AGEGLNSQFWSLAAPQRF (SEQ ID NO: 44)Neuropeptide SF human (NPSF) SQAFLFQPQRF (SEQ ID NO: 45) Substrate PRPKPQQFFGLM (SEQ ID NO: 46) β-human-Casomorphin YPFVEPIP (SEQ ID NO: 47)β-bovine-Casomorphin YPFPGPI (SEQ ID NO: 48)

Of the above opioids β-endorphins, enkephalins, and dynorphin are threehuman endogenous opioids. β-Endorphins are primarily found in thearcuate nucleus of the hypothalamus and in the pituitary gland, afeature that distinguishes this group from the enkephalins, which arenot present in that area. Enkephalins may be broken down into two types,methionoine enkephalin (met-enkephalin) and leucine enkephalin(leu-enkephalin), and their ratio is 4:1 respectively. They are morewidely distributed in the brain than β-endorphins, being present inseveral areas including hypothalamic nuclei, limbic structures,caudate-putamen, the brain stem, several layers of the dorsal horn,peripheral nerves, and the adrenal medulla. The most powerful of theopioids, dynorphins, are found throughout the central and peripheralnervous systems. Some research supports the theory that they regulatepain at the spinal cord level, influence feeding behavior at thehypothalamic level, and function with other endogenous opioids toregulate the cardiovascular system. Dynorphins also may be involved ininhibiting intestinal motility, a phenomena that occurs when the bodyperceives pain. The presence of a large precursor to this opioid in theanterior pituitary suggests that it has many peripheral targets. Anotheropioid called neo-endorphin also is classified in the Dynorphin group.

The endogenous opioid system has been used to treat chronic pain througha technique called neuroaugmentation that involves electricalstimulation of specific areas of the brain to increase the quantity andreactivity of endogenous opioids. Partial or complete pain relief hasbeen noted in patients treated with neuroaugmentation; lower levels ofefficacy were observed in severely ill cancer patients. Spinal cordstimulation was found to be successful in treating chronic pain notassociated with malignancy.

In some embodiments, the present invention contemplates the use of anyof the opioids herein (whether naturally occurring or not) formodulating heat production, innate immune mechanisms, or mitochondrialactivity in plants and/or animals as described in more detail herein.

2. Temperature Regulation

In one aspect, the compositions herein can also be used to regulate bodytemperature of an animal or a plant by modulating the outflow and inflowof heat from the body. Fever, an elevated core body temperature, is themost common thermoregulatory change that often accompanies inflammationand/or infection. One main source of heat in both plants and animals isthe mitochondria. Another major source of heat in animals is muscularcontraction. However, in plants, it is often difficult to measure theirbody/appendix temperature because the amount of heat produced isrelatively small and the there is a large amount of heat loss to theenvironment.

In humans, opioids that have been used to alleviate pain have beenlinked to some degree with temperature regulation in humans. Inparticular, there is some evidence that suggests a link between thenervous system and thermoregulation (Thermoregulation, TenthInternational Symposium on the Pharmacology of Thermoregulation.Blatteis C M, Ed., Annal. NY Acad. Sci. vol., 813, 1997), and morespecifically between opioid peptides and thermoregulation (Adler M W etal, 1988, Opioid System and Temperature Regulation, Annu. Rev.Pharmacol. Toxicol., Vol. 28: 429-450).

In one aspect, the present invention relates to the surprising discoverythat analgesics, such as opioids useful in treating pain in humans, canalso be used to modulate heat production in plants. Thus, the presentinvention relates to uses of any of the compositions herein includingfor example (i) polypeptides comprising, consisting essentially of, orconsisting of, an amino acid sequence of SEQ ID NO: 1-244, 248-249, orSEQ ID NO: 1-14 and 50-244, or SEQ ID NO: 1-2 or SEQ ID NO: 1; (ii)nucleic acids encoding any of the above polypeptides; and (iii)antibodies that specifically bind any of the above polypeptide, formodulating heat production in plants (e.g., crops or flowering plants).

Such heat production modulation can be used e.g., to prevent frostdamage to the seed and plants. It can also increase the innate immuneresponse of the plants (discussed in more detail below). The above usescan be accomplished by administering to a plant or seed any of the abovecompositions with an agricultural excipient via spray, drip irrigationor other irrigation, dipping at least a portion of said plant or seed insaid composition, coating at least partially said plant or seed withsaid composition, etc. In another embodiment, a nucleic acid sequenceencoding any of the above compositions can be used to transfect plantssuch that their heat production is regulated.

In another aspect, the present invention relates to methods of using theabove compositions for modulating mitochondrial activity in plants andanimals. Modulating mitochondrial activity can be used to prevent,treat, or ameliorate mitochondrial conditions in plants and animals.

Examples of mitochondrial conditions include, but are not limited to,Alpers disease (progressive infantile poliodystrophy); Barth syndrome(cardiomyopathy-neutropenia syndrome); lethal infantile cardiomyopathy(LIC); Beta-oxidation defects; carnitine deficiency and disorders;chronic progressive external ophthalmoplegia syndrome (CPEO);Kearns-Sayre syndrome (KSS); lactic acidosis; Leber hereditary opticneuropathy (LHON); Leigh disease (subacute necrotizingencephalomyelopathy); long-chain acyl-CoA dehydrogenase deficiency(LCAD); Luft disease; medium-chain acyl-CoA dehydrogenase deficiency(MCAD); mitochondrial cytopathy; mitochondrial encephalomyopathy lacticacidosis and stroke-like episodes (MELAS); mitochondrial encephalopathy;mitochondrial myopathy; multiple acyl-CoA dehydrogenase deficiency(MAD); glutaric aciduria Type II; myoclonic epilepsy and ragged-redfiber disease (MERRF); myoneurogastointestinal disorder andencephalopathy (MNGIE); neuropathy ataxia and retinitis pigmentosa(NARP); pearson syndrome; pyruvate carboxylase deficiency; pyruvatedehydrogenase deficiency (PHD); and short-chain acyl-CoA dehydrogenasedeficiency (SCAD).

Other examples of mitochondrial conditions include respiratory chaindisorders such as: Complex I: NADH dehydrogenase (NADH-CoQ reductase)deficiency; Complex II: Succinate dehydrogenase deficiency; Complex III:ubiquinone-cytochrome c oxidoreductase deficiency; Complex IV:cytochrome c oxidase (COX) deficiency; and Complex V: ATP synthasedeficiency.

In one embodiment, an organism such as a plant or animal can bediagnosed for the presence of a mitochondrial condition by geneticallyscreening the organism. In some embodiments, the organisms' genetic DNAor mitochondrial DNA can be analyzed. In some embodiments, theorganism's RNA, mRNA, siRNA, or cRNA is analyzed.

An organism having by or susceptible of having a mitochondrial conditioncan then be administered one or more of the compositions disclosedherein to modulate, treat, or prevent the condition. Such compositionsinclude, but are not limited to (i) a polypeptide comprising an aminoacid sequence selected from the group consisting of: SEQ ID NO: 1-14 and50-244 or an analog, homolog, mimetic or salt thereof; (ii) a nucleicacid sequence encoding at least in part a polypeptide comprising anamino acid sequence selected from the group consisting of: SEQ ID NO:1-14 and 50-244; and (iii) an antibody or antibody fragment thatspecifically binds a polypeptide comprising an amino acid sequenceselected from the group consisting of: SEQ ID NO: 1-14 and 50-244.

3. Inflammation

Acute and chronic pain is frequently associated with inflammation as aresult of tissue destruction, abnormal immune reactivity or nerveinjury.

In one aspect, the compositions herein can also be used to treat,modulate, or prevent inflammation in an organism. The inflammation canbe due to a variety of external or internal insults, such as infectiousagents, physical injury, hypoxia, or disease processes in nearly anyorgan or tissue in the body with one or more of the following symptoms:redness, heat, tenderness/pain, and swelling. Other examples areinflammatory diseases which the compositions herein can be used to treatinclude those such as rheumatoid arthritis, inflammatory bowel disease,scleroderma, cutaneous lupus erythematosus, systemic lupuserythematosus, type 1 and II diabetes, asthma, multiple sclerosis,abscess, wounds, meningitis, encephalitis, vasculitis, andcardiovascular diseases.

Since the discovery of salicylic acid (SA) as an anti-inflammatorycompound and the subsequent synthesis of aspirin (ASA) over a centuryago, several classes of structurally diverse compounds have becomeavailable for the treatment of human inflammatory disorders. Thesecompounds are collectively known as NSAIDs and share with ASA a commonmechanism by which they exert their anti-inflammatory action.Inflammation is now recognized as a type of immune response that directsimmune system components to the site of injury or infection and is amajor contributor to many diseases. Inflammation can be localized to awound or an injury site and it can be systemic. Recent studies show apossible link between cardiovascular diseases and inflammation, e.g.,the levels of C-reactive polypeptide, a molecular marker ofinflammation, rank with cholesterol levels as indicators of futurecoronary heart disease.

In one aspect, the present invention relates to the use of thecompositions herein including for example (i) polypeptides comprising,consisting essentially of, or consisting of, an amino acid sequence ofSEQ ID NO: 1-151, or more preferably SEQ ID NO: 1-14, 50-244, 248-249,or more preferably SEQ ID NO: 1-2, or more preferably SEQ ID NO: 1; (ii)nucleic acids encoding any of the above polypeptides; and (iii)antibodies that specifically bind any of the above polypeptide, for thetreatment of inflammatory conditions.

In some embodiments, the compositions above can be used in combinationwith one or more other anti-inflammatory agents to relief theinflammation.

4. Neoplastic Growth

Cell division and growth are essential for development and repair oforgans and tissues. However excess or uncontrolled growth is importantcauses of disease such as cancer. Endogenous opioid peptides have playeda role in regulating immunity and tumor growth. In addition to their usein the treatment of pain, opioids, appears to be important in the growthregulation of normal and neoplastic tissue (Rasmussen et al., 2002, NEL.23:193-198). For example, release of endogenous opioids has been foundto stimulate growth of 1 breast cancer in rats and opiate receptorantagonists have reduced the growth of these tumors (Balslev et al.,1989, Am. J. Path., 134:473-479). In another example, cyclooxygenase-2(COX-2) and the prostaglandins resulting from its enzymatic activityhave also been shown to play a role in modulating cell growth anddevelopment of human neoplasia. Evidence includes a direct relationshipbetween COX-2 expression and cancer incidence in humans and animalmodels, increased tumorigenesis after genetic manipulation of COX-2, andsignificant anti-tumor properties of NSAIDs in animal models and in somehuman cancers. Moreover, recent data showed that COX-2 and the derivedprostaglandins are involved in control of cellular growth, apoptosis,and signal through a group of nuclear receptors named peroxisomeproliferator-activated receptors (PPARs; Trifan and Hla, 2003, J. Cell.Mol. Med. 7:207-222; Martinsgreen et al., 1994, Cancer Res.54:4334-4341).

Thus, any of the compositions herein can also be used for the treat,prevent or modulate aberrant cell growth and in particular, cancer.

Non-limiting examples of cancers that may be modulated, treated, orprevented by the compositions herein include, but are not limited to,breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer,lung cancer, brain cancer, cancer of the larynx, gallbladder, pancreas,rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck,colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cellcarcinoma of both ulcerating and papillary type, metastatic skincarcinoma, osteo sarcoma, Ewing's sarcoma, reticulum cell sarcoma,myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet celltumor, primary brain tumor, acute and chronic lymphocytic andgranulocytic tumors, hairy-cell leukemia, adenoma, hyperplasia,medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinalganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitustumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor,cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma,soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosisfungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and othersarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforme, leukemias, lymphomas, malignantmelanomas, epidermoid carcinomas, and other carcinomas and sarcomas.

The largest class of tumors falls into the ectoderm/endoderm class. Thisclass includes the leading causes of death in humans (bronchogeniccarcinoma, colon adenocarcinoma, breast carcinoma and prostate carcinomaand the most frequently occurring (though usually non-lethal) tumors ofhumans (squamous cell carcinoma of skin and basal cell carcinoma ofskin). The other tumor groups are tumors of mesodermal lineage(including all sarcomas) and tumors of neuroectodermal lineage.

Thus, in some embodiments, a composition herein (e.g., SEQ ID NO: 1) canbe administered to a subject susceptible of or having cancer to treat,modulate, or prevent the condition. Such compositions include forexample (i) polypeptides comprising, consisting essentially of, orconsisting of, an amino acid sequence of SEQ ID NO: 1-244, 248-249, ormore preferably SEQ ID NO: 1-14 and 50-244, or more preferably SEQ IDNO: 1-2, or more preferably SEQ ID NO: 1; (ii) nucleic acids encodingany of the above polypeptides; and (iii) antibodies that specificallybind any of the above polypeptide, for the treatment of inflammatoryconditions.

Such compositions can be administered along with one or moreanti-neoplastic agents or be co-formulated with one or moreanti-neoplastic agents to increase their therapeutic effect.

Anti-neoplastic agents can be grouped into the following generalcategories: alkylating agents, anti-metabolites, mitotic inhibitors,anti-neoplastic antibiotics, hormonal agents, and miscellaneous. Examplefor an alkylating agent is Mechlorethamine hydrochloride that is used totreat Hodgkin's disease in man. Example for antimetabolites ismethotrexate, an inhibitor of dihydrofolate reductase. Examples formitotic inhibitors are Paclitaxel and docetaxel that are antimicrotubuleagents. Examples for antineoplastic antibiotics are Mitoxantrone, ananthracenedione related to the anthracycline antibiotics, Doxorubicinand Bleomycin. Examples for hormonal agents are glucocorticoids.

Additional examples of antineoplastic agents include, but are notlimited to: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine;Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate;Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase;Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa;Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin;Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan;Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin;Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol;Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; DaunorubicinHydrochloride; Decitabine; Dexormaplatin; Dezaguanine; DezaguanineMesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride;Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin;Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin;Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole;Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium;Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate;Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine;Fludarabine Phosphate; Fluorouracil; Fluorocitabine; Fosquidone;Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198;Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Imofosine; InterferonAlfa-2a; Interferon Alfa-2b; Interferon Alfa-n1; Interferon Alfa-n3;Interferon Beta-Ia; Interferon Gamma-Ib; Iproplatin; IrinotecanHydrochloride; Lanreotide Acetate; Letrozole; Leuprolide AcetateLiarozole Hydrochloride; Lometrexol Sodium; Lomustine; LosoxantroneHydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride;Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril;Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine;Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin;Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; MycophenolicAcid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel;Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin1, SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid;Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin;Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine;Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; ToremifeneCitrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate;Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; UracilMustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate;Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate;Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate;Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin;Zinostatin; and Zorubicin Hydrochloride.

5. Innate Immune System

An organism has inborn defense mechanisms or innate immune system thatallows it to defend itself against invasions by pathogens. Thecompositions herein can also be used to modulate, prevent and/or treatpathogen invasions (bacteria, virus, and fungi, crop pests, etc.) inhumans and plants.

In humans, the few microbes that manage to cross the barriers of skin,mucus, cilia, and pH are usually eliminated by innate immune system,which commence immediately upon pathogen entry. If phagocytosis cannotrapidly eliminate pathogen, inflammation is induced with the synthesisof cytokines and acute phase polypeptides. This early-induced responseis not antigen-specific. Only if the inflammatory process isunsuccessful at eliminating pathogen, the adaptive immune system isactivated.

Plants also possess the mechanism of self-defense against pathogens andother abiotic stresses. (Cohen et al., (2001) Curr. Opin. Immunol13:55-62) Salicylic acid plays an important role in the induction ofresistance to a broad spectrum of widely different pathogens such asfungi, bacteria or viruses such as the bacteria Pseudomonas syringae andthe tobacco mosaic virus (TMV). While conventional pesticides targetsthe pathogens, most non-conventional pest control chemicals(biopesticides) are based on small molecule production either by addedgenetic material or microorganisms, which increases a plants ability tofight pathogens.

Thus, in some embodiments, the compositions herein are used to increasea seed, plant (e.g., crop) or plant cuttings' innate immune response topathogen (e.g., bacteria, viruses, fungi, crop pests). This can helpreduce crop losses. Other examples of conditions that may result in croplosses that can be preventable or diminished by the compositions hereininclude stress conditions such as drought, freezing or reducedtemperatures, and other unfavorable environmental conditions (seediscussion of temperature regulation above).

Examples of plants that may be treated with the compositions hereininclude, culture plans such as wheat, barley, rye, oats, rice, sorghumand the like; including Chenopodiaceae, e.g., sugar beet and fodderbeet; pome and stone fruits and berries, e.g., apples, pears, plums,peaches, almonds, cherries, strawberries, raspberries and blackberries;Legume, e.g., beans, lentils, peas, soy beans; Brassicaceae, e.g., rape,mustard, cabbages and turnips. Cucurbitaceae, e.g., pumpkins, gherkins,melons, cucumbers, squashes; fibrous plants, e.g., cotton, flax, hemp,jute; citrus fruits, e.g., orange, lemon, grapefruit, mandarin;vegetables, e.g., spinach, lettuce, asparagus, ground-nuts; carrots,onions, tomatoes, potatoes, hot and sweet peppers; laurel-like plants,e.g., avocado, cinnamon, camphor tree; or plants such as maize, tobacco,nuts, coffee, sugar-cane, tea, vines, hops, bananas, rubber plants,poppy, olive, sunflower, coconut, castor-oil plant, cocoa as well asornamental plants, e.g., flowers, shrubs, deciduous trees and evergreentrees such as conifers. This list is given with the purpose ofillustrating the invention and not to delimiting it thereto.

Thus, in some embodiments, a composition herein can be administered to aplant or animal to prevent or treat a pathogen invasion. Suchcompositions include for example (i) polypeptides comprising, consistingessentially of, or consisting of, an amino acid sequence of SEQ ID NOs:1-244, 248-249, or more preferably SEQ ID NOs: 1-14 and 50-244, or morepreferably SEQ ID NOs: 1-2, or more preferably SEQ ID NO: 1; (ii)nucleic acids encoding any of the above polypeptides; and (iii)antibodies that specifically bind any of the above polypeptide, for thetreatment of inflammatory conditions. Such compositions can furtherinclude a veterinary excipient, pharmaceutical excipient or agriculturalexcipient.

6. Neurological Condition

Examples of neurological and neurodegenerative conditions that may bemodulated, treated, or prevented by the compositions herein include, butare not limited to, anxiety disorder, panic disorder,obsessive-compulsive disorder, post-traumatic stress disorder, socialphobia (or social anxiety disorder), specific phobias, and generalizedanxiety disorder. Any of the above conditions can also be accompanied byor manifested by other conditions such as depression, drug abuse,alcoholism, Aicardi syndrome, Alzheimer's disease, amnesia, amyotrophiclateral sclerosis (Lou Gehrig's disease), anencephaly, aphasia,arachnoiditis, Arnold Chiari malformation, Batten disease, Bell's Palsy,brachial plexus injury, brain injury, brain tumors, Charcol-Marie toothdisease, dystonia, encephalitis, epilepsy, essential tremor,Guillain-Barre syndrome, hydrocephalus, hyperhidrosis, Krabbes disease,leukodystrophy, meningitis, Moebius syndrome, multiple sclerosis,muscular dystrophy, Parkinson's disease, peripheral neuropathy, posturalorthostatic tachycardia syndrome, progressive supranuclear palsy,prosopagnosia, shingles, Shy-Drager syndrome, spasmodic torticollis,spina bifida, spinal muscular atrophy, stiff man syndrome, synesthesia,syringomyelia, thoracic outlet syndrome, tourette syndrome,toxoplasmosis, and trigeminal neurolagia.

V. Screening Assays

As used herein, the term “screening” refers to the use of assaysdesigned to identify agents that alter (e.g., increase or decrease in astatistically significant manner) one or more of the compositions herein(e.g., a polypeptide comprising, consisting essentially of, orconsisting of an amino acid sequence of (or encoded by) SEQ ID NOs:1-244, 248-249, or any of the above with a methionine at theN-terminus). Briefly, in certain embodiments, when the compositionsherein are contacted with a known polypeptide in the absence andpresence of a candidate agent and under conditions and for a timesufficient for binding to the polypeptide to occur, and the effect ofthe agent on the binding interaction between the polypeptide and acomposition herein is determined A candidate agent may alter any of theherein described parameters directly (e.g., by physical contact with thepolypeptide at a site of ligand binding) or indirectly (e.g., byinteraction with one or more proximal or distal sites within thepolypeptide, as may according to non-limiting theory alter the describedparameter by interacting with other than a site of ligand binding, forinstance, electron transfer or UV absorbance, or changing theconformation of the polypeptide. In some embodiments, the candidateagent may be a peptide, polypeptide, polypeptide or small molecules, andin certain preferred embodiments the candidate agent may be a structuralmimetic of one or more of the compositions herein. Typically, and inmore preferred embodiments such as for high throughput screening,candidate agents are provided as “libraries” or collections ofcompounds, compositions or molecules. Such molecules typically includecompounds known in the art as “small molecules” and having molecularweights less than 10⁴ daltons, preferably less than 10⁵ daltons. Forexample, members of a library of test compounds can be administered to aplurality of samples, each containing at least one homolog of apolypeptide herein and a known polypeptide as provided herein, and thenassayed for their ability to alter at least one of the above-describedparameters.

The present invention provides compositions, methods and kits for use in(i) identifying agents that alter a biological effect by thecompositions herein, and (ii) identifying peptides and peptidomimeticsthat function like the compositions herein.

One or more of above peptides can be used to screen small molecules andother compounds (e.g., antibodies, peptides, peptide nucleic acids, andnucleic acids) that interact with SEQ ID NOs: 245-247, 250, 253-256.Such library of compounds can include at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 40, 50, 60 or 70 of the polypeptides herein.

Candidate agents further may be provided as members of a combinatoriallibrary, which preferably includes synthetic agents prepared accordingto a plurality of predetermined chemical reactions performed in aplurality of reaction vessels. For example, various starting compoundsmay be prepared employing one or more of solid-phase synthesis, recordedrandom mix methodologies and recorded reaction split techniques thatpermit a given constituent to traceably undergo a plurality ofpermutations and/or combinations of reaction conditions. The resultingproducts comprise a library that can be screened followed by iterativeselection and synthesis procedures, such as a synthetic combinatoriallibrary of peptides. Those having ordinary skill in the art willappreciate that a diverse assortment of such libraries may be preparedaccording to established procedures, and tested using the knownpolypeptides as a target.

There are a variety of assay formats known to those of ordinary skill inthe art for detecting binding interactions between polypeptides andtheir cognate ligands. See, e.g., Harlow and Lane, 1988 In: Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory. Within oneembodiment, a polypeptide or polypeptide is immobilized on a solidsupport prior to contact with the ligand. Binding may then be detectedusing a detection reagent that specifically binds to the polypeptide,for example, at a site known or suspected of being a site of ligandinteraction (e.g., an antibody or fragment thereof), or using adetectable portion of the polypeptide (e.g., direct detection of aUV-absorbing moiety, or detection of electron transfer to an acceptormolecule).

A solid support may be any material known to those of ordinary skill inthe art. For example, the solid support may be a test well in amicrotiter plate or a nitrocellulose or other suitable membrane.Alternatively, the support may be a bead or disc, such as glass,fiberglass, latex or a plastic material such as polystyrene orpolyvinylchloride. A polypeptide may be immobilized on the solid supportusing a variety of techniques known to those of skill in the art, whichare amply described in the patent and scientific literature. In thecontext of the present invention, the term “immobilization” refers toboth non-covalent association, such as adsorption, and covalentattachment (which may be a direct linkage between the agent andfunctional groups on the support or may be a linkage by way of across-linking agent). Immobilization by adsorption to a well in amicrotiter plate or to a membrane is preferred. In such cases,adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.

Binding is generally allowed to occur under solution conditions and foran amount of time sufficient to detect the bound ligand. An appropriateamount of time may generally be determined by assaying the level ofbinding that occurs over a period of time. After incubating underconditions and for a time sufficient to permit interaction of apolypeptide of the invention and candidate receptor agent, the level ofthe ligand-receptor binding is detected and compared to the level ofbinding in the presence and absence the polypeptide of the invention.

For example, following a suitable interval for competitive ligandbinding, unbound ligand is removed, and bound ligand is detected using alinked reporter group or a separate detectable marker comprising areporter group. The method employed for detecting binding depends uponthe nature of the reporter group employed. When electron transfer isdetected, fluorescence or colorimetric or other techniques may be used.For radiometric quantification of ligand binding (or, e.g., competitiveinhibition by a candidate agent of the binding site for a polypeptidecomprising, consisting essentially of, or consisting of an amino acidsequence of (or encoded by) SEQ ID NOs: 1-244, 248-249, or any of theabove with a methionine at the N-terminus, of a detectably labeledligand comprising a radioactive group), scintillation counting orauto-radiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

An agent that binds to a polypeptide of the invention and/or to apolypeptide complex comprising a polypeptide of the invention may resultin a detectable decrease or increase in binding the polypeptide to itsnatural receptor. Such altered levels of ligand-receptor binding can bedetected by a statistically significant increase or decrease in bindingto the receptor. Such agents that interfere with the ligand-receptorbinding may be used as inhibitors of the compositions herein.

One or more of above peptides can be used to screen small molecules andother compounds (e.g., antibodies, peptides, peptide nucleic acids, andnucleic acids) that interact with SEQ ID NOs: 245-247, 250, 253-256.Such library of compounds can include at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 40, 50, 60 or 70 of the polypeptides herein.

The present invention provides compositions, methods and kits for use ina phage display peptide library in which a library of variants of apeptide SEQ ID NOs: 1-244, 248-249, is expressed on the outside of aphage virion, and the DNA encoding each variant resides inside thevirus. This creates a physical linkage between each variant proteinsequence and the DNA encoding it, which allows rapid partitioning basedon binding affinity to a given target molecule (antibodies, enzymes,cell-surface receptors, etc.) by an in vitro selection process calledpanning (Whaley et al., 2000, Nature 405:665-668).

The present invention also provides methods for identifying leadcompounds for treatment of mitochondrial and metabolic conditions suchas pain, inflammation, fever, Alzheimer's disease and any other diseasementioned herein. Such methods involve the use of a thermogenic plantfor studying thermoregulation activity by candidate agents. Examples ofthermogenic plants include those such as the Sauromatum guttatum,members of the Araceae family, Amorphophallus konjac, Arum italicum, A.dioscoridis, Dracunculus vulgaris; lotus (Nelumbonaceae), Dutchman'spipes (Aristolochiaceae), palms (Arecaceae and Cyclanthaceae), custardapples (Annonaceae), magnolias (Magnoliaceae), Illicium (Illiciaceae),Rafflesia (Rafflesiaceae), winter's bark (Winteraceae) and cycads(Cycadaceae).

In one example, using Sauromatum guttatum as the experimental module, onthe day of inflorescence-opening, the Sauromatum appendix (a 20-40cm-long, slender organ) becomes warm, reaching a 32° C. temperature(Skubatz et al., 1991, Plant Physiol. 95:1084-1088). The heat generatedby the appendix is generated by the mitochondria. This mitochondrialactivity can be triggered by the addition of phenolic compounds,including but not limited to, salicylic acid, aspirin, and2,6-dihydroxybenzoic acid. Test agents can be applied to the plant andthe plant's temperature may be monitored in vivo.

Test agents (e.g., polypeptide, such as those disclosed herein) that canmodule (increase, decrease, sustain, or shorten) heat generated by theplants can be used as lead compounds in animal models to test theirability to treat/modulate a metabolic condition. In some embodiments,small molecules or mimetics of the test compounds are applied to theplants. A small molecule or mimetic that has the same effect as a testagent such as a polypeptide may be then tested in an animal model forits ability to treat/modulate a metabolic condition. In someembodiments, such test compounds are agonists, antagonists and/or othermodulators of mitochondrial activities.

Administration

The compositions (including formulations) herein can be administeredsystemically or locally to a plant or animal by any means known in theart. For example, to an animal such as a human, the compositions hereincan be administered parenterally (which includes subcutaneously,intravenously, intramuscularly, intrasternally, intracavernously,intrathecally, and intraurethrally), intracranially, intraorbitally,intracapsularly, intraspinally, intracistemally, intrapulmonaryl), (viainhallation), orally, intravenously, intra-arterially, intramedullary,intrathecally, intraventricularly, intrameatally, transdermally,subcutaneously, intraperitoneally, intranasally, enterally, vaginally,sublingually, or rectally. Preferably, the compositions herein areadministered to an animal topically, subdermally or intravenously. Insome embodiments, the composition/formulations herein are administeredusing insert(s), bead(s), timed-release formulation(s), patch(es) orfast-release formulation(s).

For plants, the compositions herein can be administered by any methodknown in the art, including, but not limited to, spray, drip irrigationor other irrigation, dipping at least a portion of said plant or seed insaid composition, coating at least partially said plant or seed withsaid composition, etc. In another embodiment, a nucleic acid sequenceencoding any of the above compositions can be used to transfect plantssuch that their heat production is regulated.

The compositions/formulations herein are preferably administered in aneffective dose. It will be evident to those skilled in the art that thenumber, frequency, and duration of administration will be dependent uponthe response of the host.

For therapeutic delivery, agents at concentrations of about 0.01 μg/kgto about 1000 mg/kg body weight may be administered, typically by theintradermal, subcutaneous, intramuscular or intravenous route, or byother routes. A preferred dosage is about 1 μg/kg to about 1000 mg/kg,or about 5 μg/kg to about 500 mg/kg, or about 10 μg/kg to about 100mg/kg.

For agricultural delivery, agents may be administered at a concentrationthat is agriculturally therapeutically effective, e.g., about 50-3000gram per hectare, preferably from about 50-1500 gram per hectare, andmore preferably from about 150-300 gram per hectare. Assuming acomposition is comprised of 100% active ingredients, then, in general,the amount of the subject composition used will range from about 0.005%to 25% of the weight of the seed, and more preferably, from about 0.01%to about 10% of the weight of the seed. In yet another embodiment theamount of the subject composition used will be in the range of 0.01% to1% of the active ingredients relative to the weight of the seed, or0.05% to 0.5%.

EXAMPLES Example 1

A total of forty (40) male Sprague Dawley rats (Harlan Sprague DawleyInc., Indianapolis, Ind., USA) were used in the study. The rats arespecific pathogen free and approximately 250 grams upon arrival. Therats were housed in the vivarium in clear polycarbonate plastic cages(48×27×20 cm); 2 rats per cage until a few days prior to surgery atwhich point they were singularly housed for the remainder of the studyprocedures. The bedding material is irradiated corn-cob bedding(Bed-O-Cob, The Andersons, Maumee, Ohio, USA) that was changed weekly.The rats were acclimated for two weeks prior to the commencement of theexperimental procedures. The room in which the rats were housedthroughout the study was supplied with HEPA filtered air at the rate of10-15 air changes per hour. The temperature was maintained at 18-26° C.with a relative humidity of 30-70%. Illumination is approximately 300lumens/m² at 1 m above floor level on a 12-hour light/dark cycle. Therats had ad libitum access to oval pellet Certified Picolab Rodent Diet20 (PMI Feeds Inc., Richmond, Ind., USA) and deionized water.

All rats were anesthetized with Inhalation anesthetic (Isoflurane). Theplantar aspect of the foot was cleaned and prepped for aseptic surgery.The animals were placed in ventral recumbancy. A 1-246 cm longitudinalincision was made with a #11 blade, through the skin and fascia of theplantar aspect of the foot, starting 0.5 cm from the proximal edge ofthe heel and extending towards the toes. The plantaris muscle waselevated and incised longitudinally. The muscle origin and insertionremained intact. Gentle pressure was applied for hemostatis, if needed.The skin was closed with suture material and the wound site covered witha triple antibiotic ointment. The rats were allowed to recover in theircage until regaining full mobility.

On days 1 and 3 post-surgery, animals were dosed with the appropriatetest or control compound (Table III).

TABLE III Treatment groups for the first study of the analgesic propertyof SEQ ID NO: 1 on post-operative pain in rats. Group No. SurgeryTreatment Dose Route 1 Sham (No Vehicle 0.1 mL Topical surgery) 0.17%DMSO, 0.05% Silwet in H2O 2 Brennan model Vehicle 0.1 mL Topical 3Brennan model Morphine in PBS   5 mg/kg Subcutaneous 4 Brennan model SEQID NO: 1 in vehicle 150 μg/0.15 mL Topical 5 Brennan model SEQ ID NO: 1in PBS 150 μg/0.1 mL Subdermal

On days 1 and 3 post-surgery, the rats underwent Von Frey testing formechanical allodynia. Tactile sensitivity (i.e. mechanical allodynia)was measured using calibrated filaments touched to the plantar surfaceof the affected limb.

Procedurally, the rats were placed in a plastic cage with a wire meshbottom and allowed to acclimate for 5 to 10 minutes. Once the animalssettled, the plantar surface of the right hind paw was touched with a2.0 g von Frey filaments. In the absence of a paw withdrawal response tothe initially selected filament, a stronger stimulus was presented; inthe event of paw withdrawal, the next weaker stimulus was chosen. Inthis fashion, the resulting pattern of positive and negative responseswas used to determine the paw withdrawal threshold.

FIG. 1. Data were analyzed using a one-way ANOVA followed byNewman-Keuls' Multiple Comparison. Statistical significance was p<0.05.It shows that SEQ ID NO: 1 was able to significantly reduce pain.Abbreviations: TA, test article, which is SEQ ID NO: 1; sderm,sub-dermal injection; top, topical application; veh, vehicle; surg,surgery; morph, morphine. The rats appeared relaxed and with lessanxiety.

Example 2

In a second independent study the rat hind paw withdrawal sensitivitywas again evaluated after 3 h and 3 day post-surgery when the rats hadreceived SEQ ID NO: 1 at three different concentrations: 1, 15 and 50mg/kg. The dosing route was subdermal injection adjacent to the woundsite. The rats received one dose 3 hours after the surgery and on Day 3post-surgery, group 3 was dosed again as described in the Table IV. Painmeasurements were taken 15-20 min after application of the drug.Baseline pain behavior was measured as follows: Withdrawal responses tomechanical stimulation were determined using von Frey filaments appliedfrom underneath the cage through openings (12×12 mm in the plastic meshfloor to an area adjacent to the intended incision. Each von Freyfilament (Target force of 0.008 g to 300 g) was applied once startingwith 0.008 g filament and continuing until a withdrawal responseoccurred or 300 g force was reached. The median force producing aresponse, determined from three tests given over a 10-min period wasconsidered the withdrawal threshold.

FIG. 2 illustrates withdrawal results show that SEQ ID NO: 1 hasanalgesic and property 3 h after surgery. In the saline treated animals,the control, 3-hour post-surgery were 25.1, 4.4 and 12.7 g, indicatingsignificant hyperalgesic response immediately after surgery whichsubsided by Day-3. In the animals treated with 50 and 15 mg/kg doses ofSEQ ID NO: 1 subdermally, the pre-dose, pre-surgery responses were 30.2and 28.1 g, which were comparable to the saline treated group indicatinguniformity of the pain response in all the three groups. The withdrawalresponse in the 50 mg/kg SEQ ID NO: 1 administered on Day-1 was 9.9 gcompared to the 4.4 g for the saline group at the same time-point,indicating a 125% effect. The data expressed as mean±SE, were analyzedusing ANOVA followed by Tukey-HSD Multiple Comparison Test. Statisticalsignificance was p<0.05.

FIG. 3 Withdrawal results show that SEQ ID NO: 1 has analgesic andproperty on Day 3 post-surgery. The group, which did not receive anyfurther dosing showed a 31% increase in response time on Day 3, comparedto the saline group at the respective time point. In the group ofanimals administered with 1 mg/kg NPL/PA2 on day 1 post-surgery, the3-hour post surgery measurements showed a 39% increase in the painthreshold. On Day-3, when these animals were administered with 15.25mg/kg of SEQ ID NO: 1 subdermally, the withdrawal response was at 21.3g, compared to the withdrawal response of saline group at 12.7 g,constituting an increase of 68%. The data expressed as mean±SE, wereanalyzed using ANOVA followed by Tukey-HSD Multiple Comparison Test.Statistical significance was p<0.05.

TABLE IV Treatment groups for the first study of the analgesic propertyof SEQ ID NO: 1 on post-operative pain in rats. Number of AnimalsConcentration Group Male Route Time of Dosing (mg/kg) 1 10 saline,vehicle Day 1, 3 h after 0 surgery 2 10 subdermal Day 1, 3 h after 50mg/kg surgery 3 10 subdermal Day 1, 3 h after  1 mg/kg surgery Day 3 15mg/kg

Example 3

An adult male subject suffering from chronic pain as a result from ofmultiple fractures to both tibia and fibula in both legs and severetraumatic soft tissue damage was topically administered a compositioncontaining a polypeptide of SEQ ID NO: 1 at regions experiencing pain(mostly knees and ankles). The pharmaceutical formulation administeredcomprised of 5 μM of SEQ ID NO: 1 in 0.01% Silwet L-77. Relief wasnoticed within 15 min. after administration of a single dose of about 5μg/10 cm2 of SEQ ID NO: 1. The relief lasted for more than a week. Ascaled score of 1 to 10 was used to evaluate treatment efficacy where ascore of 10 represented a patient with sever pain discomfort andcomplete inability to walk. A scaled score of 1 represented a patientexperiencing no pain and able to freely walk or move the legs. Prior totreatment, the patient scored an 8-9 representing chronic pain anddifficulties in walking long distance. After the treatment, the patientscored a 2-3 representing a significant decrease in pain. Treatmentefficacy lasted for about 10 days.

Example 4

FIG. 4 illustrates modulation of heat generated by aspirin (ASA) inSauromatum guttatum appendix in the presence of various opioid peptidesand the Alzheimer's peptide, Aβ 1-42. One day before heat-production,sections of the appendix were placed in different aqueous solutionscontaining ASA with or without an opioid peptide. [Leu]-Enkephalin, SEQID NO: 15; Human β-Endorphin; SEQ ID NO: 19; Dynorphin A, SEQ ID NO: 21;Endomorphin 2, SEQ ID NO: 24; Neuropeptide AF, SEQ ID NO: 44;β-human-Casomorphin, SEQ ID NO: 47; Alzheimer's peptide, SEQ ID NO: 48.Sections of the appendix were placed in distilled water that was notgenerated any heat is the control (9). Temperature was recorded withthermocouples attached to the section every 5 min. The y-axis is theappendix temperature above ambient and the x-axis shows the time of theday. This figure illustrates that opioid peptides and the neurotoxicAlzheimer's peptide, Aβ 1-42 can modulate thermogenicity in plants aswell as animals and act as mitochondrial modulators.

Example 5

FIG. 5 illustrates modulation of heat generated by salicylic acid (SA)in the presence of human opioid peptides (β-Endorphin, SEQ ID NO: 19 andNueuropeptide AF, SEQ ID NO: 44) and, the Alzheimer's peptide, Aβ 1-42,SEQ ID NO: 49, and, a plant virulent bacterial pathogen (Pst DC3000).One day before heat-production, sections of the appendix were placed indifferent aqueous solutions containing salicylic acid (SA) with orwithout a peptide or with the bacterial plant pathogen, Pseudomonassyringae pv. Tomato, DC3000. Sections from the appendix were placed indistilled water that was not generated any heat is the control (water).Temperature was recorded with thermocouples attached to the sectionsevery 5 min. The y-axis is the appendix temperature above ambient andthe x-axis shows the time of the day. This figure illustrates that thesepeptides modulate thermogenicity in plants as well as animals and act asmitochondrial modulators.

Example 6

FIG. 6 illustrates modulation of heat generated by 2,6-dihydroxybenzoicacid in the presence of the Alzheimer's peptide, Aβ1-42, SEQ ID NO: 49and a plant gene derived sequence in the Sauromatum guttatum appendix.One day before heat-production, sections of the appendix were placed indifferent solutions containing 2,6-dihydroxybenzoic acid (2,6-DHBA) withor without SEQ ID NO: 2 (FLPSEFGVDVDR) and SEQ ID NO: 51.Heat-production was monitored with thermocouples attached to thesections. A section placed in distilled water that was not generated anyheat is the control (water). The y-axis is the appendix temperatureabove ambient and x-axis shows the time of the day. The temperature wasrecorded every 5 min. Moreover, in a second experiment, application ofSEQ ID NO: 2 up to 50 μM did not generate heat (data not shown). Thisfigure illustrates that these peptides modulate thermogenicity in plantsas well as animals and act as mitochondrial modulators.

Example 7

Administration of 10 μm of SEQ ID NO: 1 in 0.01% Silwet L-77 toArabidopsis thaliana plants induces early flowering and abundance offlowers and pods.

Example 8

Seeds of Arabidopsis ecotype Columbia (Col-0) were planted in pottingsoil. Plants were cultivated in a growth chamber with 10-h d (200 μmolm⁻² s⁻¹ at 22° C.) and 14-h night (18° C.) cycles and 80% RH. Once aweek plants were supplied with water and modified one-half strengthHoagland nutrient solution: 2 mM KNO 5 mM Ca(NO₃)₂ and trace elements,pH 7.

A virulent plant pathogen, Pst Pseudomonas syringae pv tomato, DC3000was grown at 28° C. on King's medium B containing 40 mg/L tetracycline.Plants were inoculated with 1×10⁷ cfu/ml of the pathogen in 0.01% SilwetL77 (v/v) (a surfactant) and distilled water 5 weeks after sowing. Thebacterial suspension or a control solution (0.01% Silwet L77 in water)was then sprayed on the plant once.

Disease symptoms in Arabidopsis are water-soaked, spreading lesions,sometimes surrounded by chlorotic margin that eventually lead to yieldloss and plant death.

To test the efficacy of SEQ ID NO: 15, some healthy and some diseaseplants were sprayed with salicylic acid in 0.01%. Silwet L77; somehealthy and some disease plants were sprayed with SEQ ID NO: 15 in0.01%. Silwet L77, and some healthy and some disease plants were sprayedwith salicylic acid in combination with SEQ ID NO: 15 in 0.01%. SilwetL77. A control group of some healthy and some disease plants weresprayed with water in 0.01%. Silwet L77.

Symptoms of plants were classified by the percentage of infected leavesand the severity of the infection.

It was noted that application of 20 μl salicylic acid at a concentrationof 1 mM and 20 μl SEQ ID NO: 15 to the leaves is enough to triggersystemic resistance to the virulent bacteria concentration.

TABLE V Sequence listings. SEQ ID NO Sequence   1 FLPS   2 FLPSEFGVDVDR  3 KRFLPSEFGVDVDR   4 KRFFPSEFGLDVDR   5 KRFLPSEFGFDVDH   6KRFFPSEFGNDVDK   7 KRFFPSEFGTDVDR   8 KRFLPSEFGMDPPR   9 KRFLPSEFGMDPAL 10 RRFLPSEFGLDPDH  11 KRFLPSEFGMDPDI  12 KRFFPSEFGNDVDR  13KKFYPSEFGNDVDR  14 VKRFFPSEFGLDVDR  15 YGGFL  16 YGGFM  17 YGGFLRKYPK 18 YGGPLRKYP  19 YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE  20 YGGFMTSEKSQTPLVT 21 YGGFLRRIRPKLKWDNQ  22 YGGFLRRQFKVVT  23 YPTF  24 YPFF  25Tyr-D-Ala-Gly-N-Methyl-Phe-Gly-ol-  26 Tyr-D-Pen-Gly-Phe-D-Pen  27Tyr-D-Pen-Gly-D-Chloro-Phe-D-Pen  28 Tyr-D-Pen-Gly-Pen-Pen  29Tyr-D-Ser-Gly-Phe-Leu-Thr  30 Tyr-D-Ala-Gly-Phe-D-Leu  31Tyr-Gly-Gly-Phe-Met-Arg-Phe  32 D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-  33Ac-Arg-Tyr-Arg-Ile-Lys  34 Tyr-D-Arg-Phe-Lys  35Tyr-D-Ala-Gly-N-Methyl-Phe-Gly-ol  36 Tyr-D-Ala-Gly-Phe-D-Leu-D-Cys  37Tyr-D-Ala-Phe-Glu-Val-Val-Pro Gly-amide  38Tyr-D-Ala-Phe-Asp-Val-Val-Gly  39 Tyr-Pro-Methyl-Phe-D-Pro  40N,N-diallyl-Tyr-Aib-Aib-Phe-Leu  41 YPFP  42 FGGFTGARKSARKLANQ  43TEPGLEEVGEIEGQKQLQ  44 AGEGLNSQFWSLAAPQRF  45 SQAFLFQPQRF  46RPKPQQFFGLM  47 YPFVEPIP  48 YPFPGPI  49DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA  50 NPFLPS  51 NPYLPS  52NPWLPS  53 NPHLPS  54 DPFLPS  55 DPYLPS  56 DPWLPS  57 DPHLPS  58FLPPPPSS  59 YLPPPPSS  60 WLPPPPSS  61 HLPPPPSS  62 GIPYTY  63 SIPYTY 64 TIPYTY  65 GVPYTY  66 GIPFTY  67 NIPFTY  68 GIPFTF  69 GIPHTY  70YTIKAVDD  71 YTINAVDD  72 YTIEAVDD  73 YTINSVDD  74 YTIRAAND  75YTIKTIDD  76 FTIKAAND  77 FTIKAVDD  78 SFGVEASELYPDVKYT  79SWGLEASELYPDVKYT  80 SFGVEATALYPDVKYT  81 HAVFVNG  82 HCVFVKG  83HSVFVNG  84 HSAFVKG  85 HAAFVRG  86 HAGYIRG  87 HTAFVKG  88 HSSYVKG  89

 90 DFTSFTIDPSFG  91 CFAGLFLFVPCLGGCH  92 HCGGLCPVFLFLGAFC  93 GAFYAAFC 94 GFSPDITFSTFD  95 TLFYGAFC  96 PLSYGAFF  97 PLSYGGFY  98 GAFYGAFM  99FFAGYFLP 100 CFGGYYLP 101 CFAAYFAG 102 CFAGYFLT 103 FFAGYSLP 104YFGGYSLP 105 MFAGYFAG 106 YFAGYALP 107 WFADFFLP 108 PLFYGAFF 109PLYYGGFC 110 TGYIGKFLV 111 TGYIGKFIV 112 TGYIGKFVA 113 TGYIGKYIV 114TGYIGKYLV 115 TGYIGRHV 116 TGYLGRHV 117 TGYIGKRIV 118 TGFIGKRIV 119VLFKGIYGT 120 VIFKGIYGT 121 AVFKGIYGT 122 VIYKGIYGT 123 VLYKGIYGT 124VHRGIYGT 125 VHRGLYGT 126 TYKVDPYLESAEVGFS 127 TYKVDPYLESAELGWS 128TYKVDPYLATAEVGFS 129 TYKVDPYLESAEVGFS 130 TYKVDPYLESAELGWS 131TYKVDPYLATAEVGFS 132 KIYLIK 133 KIYLVK 134 RIYLIK 135 RIYLVK 136 KILYIK137 KVLYIK 138 KVLYIR 139 IKKEWL 140 IKNEWL 141 TKKEWL 142 LKEWI 143IKDEWL 144 IKGEWL 145 ILEEWK 146 LWEKKI 147 LWENKI 148 LWEKKT 149 IWEKL150 LWEDKI 151 LWEGKI 152 KWEELI 153 SPLF 154 RDVDVGFESPLFRK 155 PFPY156 SPLFPN 157 SPLYPN 158 SPLWPN 159 SPLHPN 160 SPLFPD 161 SPLYPD 162SPLWPD 163 SPLHPD 164 SSPPPPLF 165 SSPPPPLY 166 SSPPPPLW 167 SSPPPPLH168 GTYPIG 169 YTYPIS 170 YTYPIT 171 YTYPVG 172 YTFPIG 173 YTFPIN 174FTFPIG 175 YTHPIG 176 DDVAKITY 177 DDVANITY 178 DDVAEITY 179 DDVSNITY180 DNAARITY 181 DDITKITY 182 DNAAKITF 183 GNVFVAH 184 GKVFVCH 185GNVFVSH 186 GKVFASH 187 GVFAAH 188 GRIYGAH 189 GKVFATH 190 GKVYSSH 191GKVYSSH 192 CFAYFAG 193

194

195

196

197 PLFYGAFF 198 PLYYGGFC 199 GAFYAAFC 200

201

202

203

204 PLAYGAFY 205 PLFFDAFW 206 FFAGYFPL 207

208 VLFKGIYGT 209 VIFKGIYGT 210 AVFKGIYTG 211 VIYKGIYGT 212 VLYKGIYGT213 VHRGIYGT 214 VHRGLYGT 215 VIRKGIYGT 216 VIRKGIFGT 217 TGYIGKFLV 218TGYIGKFIV 219 TGYIGKFVA 220 TGYIGKYIV 221 TGYIGKYLV 222 TGYIGRHV 223TGYLGRHV 224 KILYIK 225 KVLYIK 226 KILYIR 227 KVLYIR 228 KIYLIR 229KIYLVK 230 RIYLVK 231 LWEKKI 232 LWENKI 233 LWEKKT 234 IWEKL 235 LWEDKI236 LWEGKI 237 KWEELI 238 IKKEWL 239 IKNEWL 240 TKKEL 241 LKEKL 242IKDEWL 243 IKGEWL 244 ILEEWK 245MDKKSRVLIVGGTGFIGKRIVKASLALGHPTYVLFRPEALSYIDKVQMLISFKQLGAKLLEASLDDHQGLVDVVKQVDVVISAVSG GLVRHHILDQLKLVEAIKEAGNIKR

EFGMDPDVVEDPLE PGNITFIDKRKVRRAIEAATIPYTYVSSNMFAGFFAGSLAQLQDAPRMMPARDKVLIYGDGNVKGVYVDEDDAGIYIVKSIDDPRTLNKTVYIRPPMNILSQKEVVEIWERLSGLSLEKIYVSEDQLLNMKDKSYVEKMARCHLYHFFIKGDLYNFEIGPNATEGTKLYPE VKYTTMDSYMERYL 246MGESKRTEKTRVLVVGATGYIGKRIVRACLAEGHETYVLQRPEIGLEIEKVQLFLSFKKLGARIVEGSFSDHQSLVSAVKLVDVVVSAMSGVHFRSHNILVQLKLVEAIKEAGNVKR

EFGMDPPR MGHALPPGRETFDQKMERQAIEAAGIPYTYVVGACFAAYFAGNLSQMVTLLPPKEKVNIYGDGNVKVVFADEDDIAKYTAKTLNDPRTLNKTVNIRPPDNVLTQLELVQIWEKLTGKELEKTNIAAQDFLANIEQMEIPHQAGIGHFYHIFYEGCLTDHEVGEDEEASSLY PDVKYKRMDDYLRMFL 247MATEKSKILVIGGTGYIGKFLVEASAKAGHSTFALVREATLSDPVKGKTVQSFKDLGVTILHGDLNDHESLVKAIKQVDVVISTVG SMQILDQTKIISAIKEAGNVKR

EFGVDVDRTSAVEPAKSA FAGKIQIRRTIEAEGIPYTYAVTGCFGGYYLPTLVQFEPGLTSPPRDKVTILGDGNAKAVINKEEDIAAYTIKAVDDPRTLNKILYIKPSNNTLSMNEIVTLWEKKIGKSLEKTHLPEEQLLKSIQESPIPINVVLSINHAVFVNGDTNISIEPSFGVEASELYPDVKYTSVDEYLSY FA 248 YGGFLRRQFKVVT 249KYPKRSSEVAGEGDGDSMGHEDLYKRYGGFLRRIRPKLKWDNQKRYGGFLRRQFKVVTRSQEDPNAYSGELFDA 250MAWQGLVLAACLLMFPSTTADCLSRCSLCAVKTQDGPKPINPLICSLQCQAALLPSEEWERCQSFSFFTPSTLGLNDKEDLGSKSV GEGPYSELAKLSGSFLKELEKSK

ISTKENTLSKSLEEKLR GLSDGFREGAESELMRDAQLNDGAMETGTLYLAEEDPKEQV KR

KRSSEVAGEGDGDSMGHEDLYKR

251 ttt ctg ccc tca 252 ttt ctg ccc tca gaa ttt gga gta gac gta  gac aga253 MVKKIANDVSNKLFPLPKGFGDFVGIEDHIKAIKSILCLESKEARIMVGIWGQSGIGKSTIGRALFSQLSSQFHHRAFITYKSTSGSDVSGMKLSWEKELLSEILGQKDIKIDHFGVVEQRLKHKKVLILLDDVDNLEFLKTLVGKAEWFGSGSRIIVITQDKQLLKAHEIDLVYEVELPSQGLALKMISQYAFGKDSPPDDFKELAFEVAELVGSLPLGLSVLGSSLKGRDKDEWVKMMPRLRNDSDDKIEETLRVGYDRLNKKNRDNVKELLEDDVGLTMLADKSLIRITPDGDIEMHNLLEKLGREIDRAKSKGNPAKRQFLTNFEDIQEVVTEKTGTETVLGIRVPPTVLFSTRPLLVINEESFKGMQIGLWSKIDLPQGLVYLPLKLKLLKWNYCPLKSLPSTFKAEYLVNLIMKYSKLEKLWEGTLPLGSLKKMDLGCSNNLKEIPDLSLAINLEELNLSKCESLVTLPSSIQNAIKLRTLYCSGVLLIDLKSLEGMCNLEYLSVDWSSMEGTQGLIYLPRKLKRLWWDYCPVKRLPSNFKAEYLVELRMENSDLEKLWDGTQPLGSLKEMYLHGSKYLKEIPDLSLAINLERLYLFGCESLVTLPSSIQNATKLINLDMRDCKKLESFPTDLNLESLEYLNLTGCPNLRNFPAIKMGCSYFEILQDRNEIEVEDCFWNKNLPAGLDYLDCLMRCMPCEFRPEYLTFLDVSGCKHEKLWEGIQIHALLDGYELAGHLDGSIETPAPTLTTNNVVSANPQYTLWKRQDRLIFSALIGAISPPVQPLVSRATKASQIWKTLTNTYAKSSYDHIKQLRTQIKQLKKGTKTIDEYVLSHTTLLDQLAILGKPMEHEEQVERILEGLPEDYKTVVDQIEGKDNTPSITEIHERLINHEAKLLSTAALSSSSLPMSANVAQQRHHNNNRNNNQNKNRTQGNTYTNNWQPSANNKSGQRPFKPYLGKCQICNVQGHSARRCPQLQAMQPSSSSSASTFTPWQPRANLAMGAPYTANNWLLDSGATHHITSDLNALALHQPYNGDDVMIADGTSLKITKTGSTFLPSNARDLTLNKVLYVPDIQKNLVSVYRLCNTNQVSVEFFPASFQVKDLNTGTLLLQGRTKDELYEWPVTNPKATALFTTPSPKTTLSSWHSRLGHPSSSILNTLISKFSLPVSVSASNKLACSDCFINKSHKLPFSISSIKSTSPLEYIFSDVWMSPILSPDNYKYYLQKSQVKSTFIAFKALVENRFQAKIRTLYSDNGGEFIALREFLVSNGISHLTSPPHTPEHNGLSERKHRHIVETGLTLLTQASVPREYWPYAFAAAVYLINRMPTPVLSMESPFQKLFGSKPNYERLRVFGCLCFPWLRPYTHNKLEERSRRCVFLGYSTQTAYLCFDVEHKRLYTSRHVVFDEASFPFSNLTSQNSLPTVTFEQSSSPLVTPILSSSSVLPSCLSSPCTVLHQQQPPVTTPNSPHSSQPTTSPAPLSPHRSTTMDFQVPQPTAPNENGPEPEAQSPPIGPLSNPTHEAFIGPLPNPNRNPTNEIEPTPAPHPKPVKPTTTTTTPNRTTVSDASHQPTAPQQNQHNMKTRAKNNIKKPNTKFSLTATLPNRSPSEPTNVTQALKDKKWRFAMSDEFDAQQRNHTWDLVPHESQLLVGCKWVFKLKYLPNGAIDKYKARLVAKGFNQQYGVDYAETFSPVIKSTTIRLVLDVAVKKDWEIKQLDVNNAFLQGTLTEEVYMAQPPGFIDKDRPTHVCRLRKAIYGLKQAPRAWYMELKQHLFNIGFVNSLSDASLFIYWSDKSSIDAVLTSLAERFSIKDPTDLHYFLGIEATRTKQGLHLMQRKYIKDLLAKHNMADAKPVLTPLPTSPKLTLHGGTKLNDASEYRSVVGSLQYLAFTRPDIAYAVNRLSQLMPQPTEDHWQAAKRVLRYLAGTSTHDWAGDSDDYVSTNAYVIYLGKNPISWSSKKQRGVARSSTESEYRAVANAASEVKWLCSLLSKLHIRLPIRPSIFCDNIGATYLCANPVFHSRMKHIAIDYHFVRNMIQSGALRVSHVSTRDQLADALTK PLSRAHFQSARFKIGVRQLPPS 254MSTSSLRRQMKNIVHNYSEAEIKVREATSNDPWGPSSSLMSEIADLTYNVVAFSEIMSMIWKRLNDHGKNWRHVYKAMTLMEYLIKTGSERVSQQCKENMYAVQTLKDFQYVDRDGKDQGVNVREKAKQLVALLRDEDRLREERAHALKTKEKLAQTATASSAAVGSGPPPEAEQAWPQSSGEEELQLQLALAMSKEEADQPPSCGPEDDVQLQLALSLSREEHDKEERIRRGDDLRLQMAIEESKRETGGKEESSLMDLADVFTTPAPPQASDPWGGPASVPTAVPVAAAASDPWGAPAVPPAADPWGGAAPTPASGDPWRPAAPTGPSVDPWGGTPAPAAGEGPTSDPWGSADGGAPVSGPPSSDPWAPAPAFSDPWGGSPAKPSSNGTAVGGFDTEPDEFSDFDRLRTALPTSGSSTGELELLAGEVPARSPGAFDMSGVGGSLAESVGSPPPAATPTPTPPTRKTPESFLGPNAALVDLDSLVSRPGPTPPGAKASNP

GAPATGPSVTNPFQPAPPATLTLNQLRLSPVPPVPGAPPTYISPL GGGPGLPPMMPPGPPAPNTNPFLL255 MEPPLPVGAQPLATVEGMEMKGPLREPCALTLAQRNGQYELIIQLHEKEQHVQDIIPINSHFRCVQEAEETLLIDIASNSGCKIRVQGDWIRERRFEIPDEEHCLKFLSAVLAAQKAQSQLLVPEQKDSSSWYQKLDTKDKPSVFSGLLGFEDNFSSMNLDKKINSQNQPTGIHREPPPPPFSVNKMLPREKEASNKEQPKVTNTMRKLFVPNTQSGQREGLIKHILAKREKEYVNIQTFRFFVGTWNVNGQSPDSGLEPWLNCDPNPPDIYCIGFQELDLSTEAFFYFESVKEQEWSMAVERGLHSKAKYKKVQLVRLVGMMLLIFARKDQCRYIRDIATETVGTGIMGKMGNKGGVAVRFVFHNTTFCIVNSHLAAHVEDFERRNQDYKDICARMSFVVPNQTLPQLNIMKHEVVIWGDLNYRLCMPDANEVKSLINKKDLQRLLKFDQLNIQRTQKKAFVDFNEGEIKFIPTYKYDSKTDRWDSSGKCRVPAWCDRILWRGTNVNQLNYRSHMELKTSDHKPVSALFHIGVKVVDERRYRKVFEDSVRIM DRMEND

LELSRREFVFENVKFRQLQKEKFQISNNGQVPCHFSFIPKLNDSQYCKPWLRAEPFEGYLEPNETVDISLDVYVSKDSVTILNSGEDKIEDILVLHLDRGKDYFLTISGNYLPSCFGTSLEALCRMKRPIREVPVTKLIDLEEDSFLEKEKSLLQMVPLDEGASERPLQVPKEIWLLVDHLFKYACHQEDLFQTPGMQEELQQIIDCLDTSIPETIPGSNHSVAEALLIFLEALPEPVICYELYQRCLDSAYDPRICRQVISQLPRCHRNVFRYLMAFLRELLKFSEYNSVNANMIATLFTSLLLRPPPNLMARQTPSDRQRAIQFLLGFLLGSEED 256MSESGNTTSMPGCGRMCALRSTWSKRAFLVACKDGALTSDGRCPQYGCGALVSITKGVQQPKKTASAKVVKCLCWVQPARWCEKHSKGPASPNGSVTTKRSNSARAAPAPLPYKKQTCDVVVTVGPLELVYPALVSEELPTPVAATPTKVEEVPIPELPLWLAPAWMVEQPYAATPEVLCLTQREEFALLKKRLTRKGKLLQRRATHARFEARAALARVRAATQRKVEEVTALVIKGRRILAAHQLLRELEEVAPLSQAQEQLVASSCAAAAARQEECASFLRRAKAWRKSISATPPVAATAVASKVVSATMPWAHLGLSLGGLLAVPTLDGTLGAKQWNAKTIATWVLKPVVSCVQSVHAKVRDWLHSQPEVGVTNTKVPLVLPEVCLGVLSPPSLSEEIVDNPQETSQSGIWHPEMGVRNIYVFHDDSWETSPEEDENYTYTFSRQCGIPYLLVEGRGAEERKNTILGWDFSLHNDGFEFLPSPEEGYTKELVTPVALEEEDKYSTASSCGFFSLDDVSSAITIQCPGLLSADADVHFFDGPGYRCSSRPRDFRPPVVRGCDYESRVKASIQRKIENPLQERFITVLREKRKKNKKKEFHSFSACFAFKRKQIQWPPTPNEMVNEWEEYCIAQAWLPFEVVVTDEIEDVTPLYPGGRDYNCNSQLLFPLAPLSTVYCDDSCFHPNDGWTTDGNGKHFRLSPQFVLPDVPIPIVHRVTRQLPQFLYDLGIGDLTCNSGYQAENLQEEIQERMEDRSEEKPVPSLDTLISKLSKRSTKVKGAGENRYADRHSLTEKAIFHQPGALSRMRSGKEKTIVAANHNSDQISVRMAECGKPVFTPLPRMSDEMLRKFLEKGLGSTSTVALDIGIQSHIPQGMPTVAFVNVMDTRIEDPLYSSLCGSYIDLGRDRAKTLCLPLVNFPMSKLAEDVDDVLNGLMLCTHFQDSTKFGVGKPAFQYGTLEFQEFKPSAYSDFSRVRDNWDAIAKQQNTPNDRILAGFSVLGAVSQAYNQALPVFKSVELVAPPKRKPVVATFQNPTTLGRSNTTRSFRMPTMDLPRSTGRDAPIPIVHRRNNNDVHFDEATPARFSTCDSGLVADTTLAFAKMYQCKKDAKAGHVLATIDIQECVFEDNRRVALDWLAHGLASFKYDLQLTVDSNPFVGVTLGITVDAFDRLLPQISDEVIAVPLAFQLPTYLFPISKKGTFTQTIDFAAIAGYNFFPHVAAFGRPKIIVYIVSDNDLPASDTWMCLVELHMTRLESSTLACSPTLVLPQAFGGDLPLDLWRGPYTFPLGGGTKRLSTSLDIGTSTTTVSGWRTVSPAAYALFLQGHGGSLVGEVVHTGSAAVSCALHLCISFGGAPPTLEEALVFPGFRLPSGEGKFHIKVQTPYGRLSTLTPDCALYVYLAGGPIAVAPMSVPYQFCIHLERLVDDGAPPRTIGLIREFNWATINNFKSDDITFAIPARLSDLVLTCGDVTMSTNPLALLIGSCGFFRGNLTVVLEWATFLKAGDKEGTVQLTTCRGMINNVKGVRNAIQKKVVNLSLVGSVSRYLNVGDFTGFAQSGGQVGYDEIFLEFSTNKAKQIRYLNINVELDENFELYGRTIIPLKNTAPAFASTSSAPNES

1. A method for identifying a small molecule or analog of SEQ ID NO: 1comprising: screening a library of candidate agents to identify acandidate agents having similar structure of function of SEQ ID NO: 1.2. The method of claim 1 wherein the screening comprises contacting theSEQ ID NO: 1 with a known polypeptide in the absence and presence of acandidate agent and under conditions and for a time sufficient forbinding of the candidate agent to the polypeptide to occur, andanalyzing effect of the candidate agent on the binding interactionbetween the known polypeptide and SEQ ID NO: 1 to identify a smallmolecule or analog of SEQ ID NO:
 1. 3. The method of claim 2 whereinfurther comprises performing high throughput screening of candidateagents.
 4. The method of claim 2 wherein a collection of candidateagents is analyzed.
 5. The method of claim 2 wherein the candidate agentis a small molecule having molecular weight of less than 10⁴ daltons. 6.The method of claim 4 wherein each member of the collection of candidateagents is administered to a plurality of samples, each sample comprisingSEQ ID NO: 1 and the known polypeptide prior to analysis.
 7. The methodof claim 2, wherein the polypeptide is immobilized on a solid support.8. The method of claim 2, wherein the candidate agent is syntheticagent.
 9. The method of claim 7, wherein the solid support is a testwell, a microtiter plate, a membrane, a bead, glass, or a disc.
 10. Themethod of claim 2 wherein the analyzing step comprises comparing levelof binding of the candidate agent to the known protein in presence andabsence of SEQ ID NO:
 1. 11. The method of claim 10 wherein level ofbinding is detected using a reagent that specifically binds to the knownpolypeptide or using a detectable portion of the known polypeptide. 12.The method of claim 2 wherein analyzing involves determining competitiveinhibition by the candidate agent of SEQ ID NOs:
 1. 13. The method ofclaim 1 wherein the screening comprises structure-based screening usingx-ray crystallography or nuclear magnetic resonance spectroscopy. 14.The method of claim 13 further comprising synthesize molecules that havesimilar or improved binding affinity to a known polypeptide.
 15. Acomposition comprising of an isolated SEQ ID NO: 344 or an analogthereof.
 16. A composition comprising an isolated SEQ ID NO: 1 or ananalog thereof.
 17. The composition of claim 15 or 16 further comprisinga pharmaceutical excipient.
 18. The composition of claim 15 or 16further comprising an agricultural excipient.
 19. A method for treatingpain in a subject comprising administering the composition of claim 15or 16.